Microbial consortium and uses thereof

ABSTRACT

Provided herein are, inter alia, microbial compositions and methods of using the same. The microbial compositions provided include, inter alia, therapeutically effective amounts of Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceus and are particularly useful for methods of treating and preventing inflammatory diseases.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT International Application No.PCT/US2017/020809, filed Mar. 3, 2017, which claims the benefit ofpriority to U.S. Provisional Application No. 62/304,087, filed Mar. 4,2016, the entire contents of each of which are hereby incorporated byreference in their entireties and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under grant numbers R21AT004732, P01 AI089473, and HL080074 awarded by the National Institutesof Health. The Government has certain rights in the invention.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The content of the text file named“48536_575C01US_Sequence_Listing.txt”, which was created on Apr. 5,2018, and is 107,578 bytes in size, is hereby incorporated by referencein its entirety.

BACKGROUND OF THE INVENTION

Recent studies provide evidence that microbial communities residing inthe human gut play a key role in the development and modulation of thehost immune response. For instance, the presence of particularClostridium species has been shown to induce specific T-cell repertoires[Atarashi, et al. (2011) Induction of colonic regulatory T cells byindigenous Clostridium species. Science 331(6015):337-341]. Despite thecomplexity of the gut microbiome, the presence or absence of specificbacterial species can dramatically alter the adaptive immuneenvironment.

BRIEF SUMMARY OF THE INVENTION

Provided herein are novel methods and microbial compositions includingLactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansiamuciniphila, Myxococcus xanthus, and Pediococcus penrtosaceus, which aresurprisingly useful for the treatment of dysbiosis, infections, andinflammatory diseases.

An aspect provides methods and compositions comprising a bacterialpopulation that comprises, consists essentially of, or consists of, 1,2, 3, 4, 5, 6, 7, or 8 (or at least 1, 2, 3, 4, 5, 6, 7, or 8) bacterialspecies. In embodiments, the bacterial population comprises, consistsessentially of, or consists of any 1, 2, 3, 4, 5, 6, 7, or 8 ofLactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcussp., Cysobacter sp., Pediococcus sp., Bifidobacterium sp., andClostridium sp. In embodiments, the bacterial population comprisesLactobacillus sp. and Faecalibacterium prausnitzii. In embodiments, thebacterial population comprises Lactobacillus sp. and Akkermansiamuciniphila. In embodiments, the bacterial population comprisesLactobacillus sp., and Myxococcus xanthus. In embodiments, the bacterialpopulation comprises Lactobacillus sp. and Cystobacter fuscus. Inembodiments, the bacterial population comprises Lactobacillus sp. andPediococcus pentosaceus, Pediococcus acidilactici, Pediococcus damnosus,Pediococcus ethanolidurans, or Pediococcus parvulus. In embodiments, thebacterial population comprises Lactobacillus sp. and Bifidobacteriumbifidum, Bifidobacterium pseudolongum, Bifidobacterium saeculare, orBifidobacterium subtile. In embodiments, the bacterial populationcomprises Lactobacillus sp. and Clostridium hiranonis. In embodiments,the Lactobacillus sp. is Lactobacillus johnsonii, Lactobacillusrhamnosus, Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillusacidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillusbrevis, Lactobacillus coleohominis, Lactobacillus crispatus,Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillusdiolivorans, Lactobacillus farraginis, Lactobacillus fermentum,Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillushelveticus, Lactobacillus hilgardii, Lactobacillus intestinalis,Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacilluskefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillusmanihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillusoligofermentans, Lactobacillus panis, Lactobacillus pantheris,Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillusparakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus,Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae,Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillussucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis. Inembodiments, the Lactobacillus sp. is Lactobacillus johnsonii. Inembodiments, the bacterial population comprises at least 1, 2, 3, 4, 5,6, 7, 8, 9, or 10, or from 1-5, 1-10, 1-5, or 1-20 of any combination ofthe following: Lactobacillus johnsonii, Lactobacillus rhamnosus,Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillusacidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillusbrevis, Lactobacillus coleohominis, Lactobacillus crispatus,Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillusdiolivorans, Lactobacillus farraginis, Lactobacillus fermentum,Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillushelveticus, Lactobacillus hilgardii. Lactobacillus intestinalis,Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacilluskefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillusmanihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillusoligofermentans, Lactobacillus panis, Lactobacillus pantheris,Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillusparakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus,Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae,Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillussucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus vini, Lactococcus garvieae, Lactococcus lactis,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus, Cystobacter fuscus, Pediococcus pentosaceus, Pediococcusacidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, andPediococcus parvulus. In embodiments, the bacterial population includesLactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansiamuciniphila, Myxococcus xanthus, and/or Pediococcus pentosaceus.

In an aspect, a method for administering isolated bacteria is provided.In embodiments, the method comprises administering to the subject aneffective amount of a bacterial population comprising Lactobacillus sp.,Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/orPediococcus sp. In embodiments, the method comprises administering tothe subject an effective amount of a bacterial population comprisingLactobacillus sp., Faecalibacterium sp., Akkermansia sp., Cystobactersp., and/or Pediococcus sp. In embodiments, the bacterial populationincludes Lactobacillus johnsonii, Faecalibacterium prausnitzii,Akkermansia muciniphila, Myxococcus xanthus, and/or Pediococcuspentosaceus. In embodiments, the bacterial population further comprisesBifidobacterium sp. or Clostridium sp. In embodiments, the bacterialpopulation further comprises Bifidobacterium sp. or Clostridiumhiranonis.

In an aspect, a method of bacterial supplementation is provided. Inembodiments, the method comprises administering to the subject aneffective amount of a bacterial population comprising Lactobacillus sp.,Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/orPediococcus sp. In embodiments, the method comprises administering tothe subject an effective amount of a bacterial population comprisingLactobacillus sp., Faecalibacterium sp., Akkermansia sp., Cystobactersp., and/or Pediococcus sp. In embodiments, the bacterial populationincludes Lactobacillus johnsonii, Faecalibacterium prausnitzii,Akkermansia muciniphila, Myxococcus xanthus, and/or Pediococcuspentosaceus. In embodiments, the bacterial population further comprisesBifidobacterium sp. or Clostridium sp. In embodiments, the bacterialpopulation further comprises Bifidobacterium sp. or Clostridiumhiranonis.

In an aspect, a method of treating or preventing inflammation in asubject in need thereof is provided. In embodiments, the methodcomprises administering to the subject an effective amount of abacterial population comprising Lactobacillus sp., Faecalibacterium sp.,Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. In embodiments,the method comprises administering to the subject an effective amount ofa bacterial population comprising Lactobacillus sp., Faecalibacteriumsp., Akkermansia sp., Cystobacter sp., and/or Pediococcus sp. Inembodiments, the bacterial population includes Lactobacillus johnsonii,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus, and/or Pediococcus pentosaceus. In embodiments, the bacterialpopulation further comprises Bifidobacterium sp. or Clostridium sp. Inembodiments, the bacterial population further comprises Bifidobacteriumsp. or Clostridium hiranonis.

In an aspect, a microbial composition is provided. In embodiments, themicrobial composition comprises Lactobacillus sp., Faecalibacterium sp.,Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. In embodiments,the microbial composition comprises Lactobacillus sp., Faecalibacteriumsp., Akkermansia sp., Cystobacter sp., and/or Pediococcus sp. Inembodiments, the composition includes Lactobacillus johnsonii,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus, Pediococcus pentosaceus and a biological carrier suitable foradministration to the gut. In embodiments, the bacterial populationfurther comprises Bifidobacterium sp. or Clostridium sp. In embodiments,the bacterial population further comprises Bifidobacterium sp. orClostridium hiranonis.

In an aspect, a microbial composition is provided. In embodiments, themicrobial composition comprises Lactobacillus sp., Faecalibacterium sp.,Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. In embodiments,the microbial composition comprises Lactobacillus sp., Faecalibacteriumsp., Akkermansia sp., Cystobacter sp., and/or Pediococcus sp. Inembodiments, the composition includes Lactobacillus johnsonii,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus or Pediococcus pentosaceus and a biological carrier suitable foradministration to the gut. In embodiments, the bacterial populationfurther comprises Bifidobacterium sp. or Clostridium sp. In embodiments,the bacterial population further comprises Bifidobacterium sp. orClostridium hiranonis.

In an aspect a pharmaceutical composition is provided. In embodiments,the pharmaceutical composition comprises Lactobacillus sp.,Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/orPediococcus sp. In embodiments, the pharmaceutical composition comprisesLactobacillus sp., Faecalibacterium sp., Akkermansia sp., Cystobactersp., and/or Pediococcus sp. In embodiments, the pharmaceuticalcomposition includes a therapeutically effective amount of Lactobacillusjohnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila,Myxococcus xanthus, and Pediococcus pentosaceus and a pharmaceuticallyacceptable excipient is provided. In embodiments, the bacterialpopulation further comprises Bifidobacterium sp. or Clostridium sp. Inembodiments, the bacterial population further comprises Bifidobacteriumsp. or Clostridium hiranonis.

In an aspect a method of treating or preventing an inflammatory diseasein a subject in need thereof is provided. In embodiments, the methodcomprises administering to the subject an effective amount of abacterial population comprising Lactobacillus sp., Faecalibacterium sp.,Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. In embodiments,the method comprises administering to the subject an effective amount ofa bacterial population comprising Lactobacillus sp., Faecalibacteriumsp., Akkermansia sp., Cystobacter sp., and/or Pediococcus sp. The methodincludes administering to the subject a therapeutically effective amountof Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansiamuciniphila, Myxococcus xanthus and Pediococcus pentosaceus. Inembodiments, the bacterial population further comprises Bifidobacteriumsp. or Clostridium sp. In embodiments, the bacterial population furthercomprises Bifidobacterium sp. or Clostridium hiranonis.

In an aspect is provided a method of increasing an anti-inflammatorymetabolite in a subject in need thereof is provided. In embodiments, themethod comprises administering to the subject an effective amount of abacterial population comprising Lactobacillus sp., Faecalibacterium sp.,Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. In embodiments,the method comprises administering to the subject an effective amount ofa bacterial population comprising Lactobacillus sp., Faecalibacteriumsp., Akkermansia sp., Cystobacter sp., and/or Pediococcus sp. Inembodiments, the method includes administering to the subject atherapeutically effective amount of Lactobacillus johnsonii,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus and Pediococcus pentosaceus. In embodiments, the bacterialpopulation further comprises Bifidobacterium sp. or Clostridium sp. Inembodiments, the bacterial population further comprises Bifidobacteriumsp. or Clostridium hiranonis.

In an aspect is provided a method of decreasing a pro-inflammatorymetabolite in a subject in need thereof is provided. In embodiments, themethod comprises administering to the subject an effective amount of abacterial population comprising Lactobacillus sp., Faecalibacterium sp.,Akkermansia sp., Myxococcus sp., and/or Pediococcus sp. In embodiments,the method comprises administering to the subject an effective amount ofa bacterial population comprising Lactobacillus sp., Faecalibacteriumsp., Akkermansia sp., Cystobacter sp., and/or Pediococcus sp. Inembodiments, the method includes administering to the subject atherapeutically effective amount of Lactobacillus johnsonii,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus and Pediococcus pentosaceus. In embodiments, the bacterialpopulation further comprises Bifidobacterium sp. or Clostridium sp. Inembodiments, the bacterial population further comprises Bifidobacteriumsp. or Clostridium hiranonis.

In an aspect a method of detecting an anti-inflammatory metabolite in asubject that has or is at risk for developing an inflammatory disease isprovided. The method includes (i) obtaining a biological sample from thesubject; and (ii) determining an expression level of ananti-inflammatory metabolite in the biological sample.

In an aspect a method of detecting a pro-inflammatory metabolite in asubject that has or is at risk for developing an inflammatory disease isprovided. The method includes (i) obtaining a biological sample from thesubject; and (ii) determining an expression level of a pro-inflammatorymetabolite in the biological sample.

In an aspect, a method of determining whether a subject has or is atrisk of developing an inflammatory disease is provided. The methodincludes (i) detecting an expression level of one or moreanti-inflammatory metabolites or pro-inflammatory metabolites in asubject; (ii) determining whether the expression level is increased ordecreased relative to a standard control, wherein an elevated expressionlevel of an pro-inflammatory metabolite or a decreased expression levelof an anti-inflammatory metabolite relative to the standard controlindicates that the subject has or is at risk of developing aninflammatory disease; and (iii) based at least in part on the expressionlevel in step (ii), determining whether the subject has or is at riskfor developing an inflammatory disease.

In an aspect, a method of determining whether a subject has or is atrisk of developing an inflammatory disease is provided. The methodincludes (i) detecting an expression level of one or morepro-inflammatory metabolites in a subject; (ii) determining whether theexpression level is increased or decreased relative to a standardcontrol, wherein an increased expression level of an pro-inflammatorymetabolite relative to the standard control indicates that the subjecthas or is at risk of developing an inflammatory disease; and (iii) basedat least in part on the expression level in step (ii), determiningwhether the subject has or is at risk for developing an inflammatorydisease.

In an aspect, a method of determining whether a subject has or is atrisk of developing an inflammatory disease is provided. The methodincludes (i) detecting an expression level of one or moreanti-inflammatory metabolites in a subject; (ii) determining whether theexpression level is increased or decreased relative to a standardcontrol, wherein a decreased expression level of an anti-inflammatorymetabolite relative to the standard control indicates that the subjecthas or is at risk of developing an inflammatory disease; and (iii) basedat least in part on the expression level in step (ii), determiningwhether the subject has or is at risk for developing an inflammatorydisease.

In an aspect, a method of monitoring the effect of treatment for aninflammatory disease in a subject undergoing inflammatory diseasetherapy or a patient that has received inflammatory disease therapy isprovided. The method includes (i) determining a first expression levelof an anti-inflammatory metabolite in the subject at a first time point;(ii) determining a second expression level of an anti-inflammatorymetabolite in the subject at a second time point; and (iii) comparingthe second expression level of an anti-inflammatory metabolite to thefirst expression level of an anti-inflammatory metabolite, therebydetermining the effect of treatment for an inflammatory disease in thesubject.

In an aspect, a method of monitoring the effect of treatment for aninflammatory disease in a subject undergoing inflammatory diseasetherapy or a patient that has received inflammatory disease therapy isprovided. The method includes (i) determining a first expression levelof a pro-inflammatory metabolite in the subject at a first time point;(ii) determining a second expression level of a pro-inflammatorymetabolite in the subject at a second time point; and (iii) comparingthe second expression level of a pro-inflammatory metabolite to thefirst expression level of a pro-inflammatory metabolite, therebydetermining the effect of treatment for an inflammatory disease in thesubject.

In an aspect, a method of determining an inflammatory disease activityin a subject is provided. The method includes (i) detecting anexpression level of one or more anti-inflammatory metabolites in asubject; (ii) determining whether the expression level is modulatedrelative to a standard control, thereby determining an inflammatorydisease activity in the subject; and (iii) based at least in part on theexpression level in step (ii), determining the inflammatory diseaseactivity in the subject.

In an aspect, a method of determining an inflammatory disease activityin a subject is provided. The method includes (i) detecting anexpression level of one or more pro-inflammatory metabolites in asubject; (ii) determining whether the expression level is modulatedrelative to a standard control, thereby determining an inflammatorydisease activity in the subject; and (iii) based at least in part on theexpression level in step (ii), determining the inflammatory diseaseactivity in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B. Significantly improved lung histology and decreased gobletcell hyperplasia is evident only in mice supplemented with C+Lj.Histological samples from three mice in each of the six experimentalgroups were stained to visualize goblet cell hyperplasia in duplicatestudies. FIG. 1A: Representative images of PAS staining for each of thesix groups clearly show that mucin secretion of goblet cells (stained)is induced in CRA challenged mice and that supplementation with L.johnsonii and microbial consortium is the only treatment group thatprotects against this induction. FIG. 1B: Image J was used to quantifythe percentage of area in each image that is positive for PAS staining.Each data point is represented by an individual symbol generated in twoindependent murine studies. Statistical analyses of this data showsignificant reductions in the percentage of PAS staining associated withthe C+Lj consortium supplemented mice compared to all other CRA treatedgroups.

FIG. 2. Significantly decreased expression of MUC5AC in micesupplemented with C+Lj supports histological findings. RNA extractedfrom the lung of each animal was used to examine the gene expressionlevel of MUC5AC, a gene involved in mucin production by goblet cells.Data from two independent murine studies is presented. Statisticalanalyses of this data show significant reductions in the percentage ofMuc5ac gene expression associated with C+Lj supplemented mice comparedto all other CRA treated groups.

FIGS. 3A-3C. Supplementation with C+Lj resulted in decreased airwayexpression of cytokines associated with allergic response. RNA extractedfrom the lung of each animal was also used to examine the geneexpression level of multiple cytokines associated with allergicresponses, including IL-4 (FIG. 3A), IL-13 (FIG. 3B), IL-10 (FIG. 3C)and IL-17. Similarly, Applicants observed a significant decrease incytokine expression associated with C+Lj supplementation for theTh2-associated cytokines (IL-4 and IL-13), as well as IL-10. Data fromtwo independent replicate studies is presented.

FIG. 4. Increased percentage of 11-17 secreting T helper cells(CD3⁺CD4⁺) is most significant in mice supplemented with C+Lj. Flowcytometry data from splenocytes reveal a significant increase in thepercentage of CD4⁺ cells expressing IL-17, a cytokine associated with Th17 cells. This observation held true across duplicated studies, and thedata from both studies are shown here.

FIGS. 5A-5B. The bacterial consortium including L. johnsonii, but not L.rhamnosus LGG, provides attenuation of allergic sensitization. The CRAallergen mouse study was performed using either L. rhamnosus LGG (LGG)or L. johnsonii (Lj) as the Lactobacillus anchor species included in thebacterial consortium supplement. To evaluate the effect of eachconsortium on sensitization, the expression level of MUC5AC wasdetermined in lung tissue using qPCR. The L. johnsonii-based consortiumsignificantly decreased MUC5AC expression, whereas the L. rhamnosus LGGconsortium failed decrease the expression of this allergic responsebiomarker.

FIGS. 6A-6C. Fecal water from a non-atopic neonate significantly reducesexpression of IL4 and IL13 responses. FIG. 6A: Fecal water exposuresignificantly decreases the number of CD4+ T-helper 2 cells in one butnot both donors. FIG. 6B: IL4 expression is significantly reduced inboth donors. FIG. 6C: IL13 expression is significantly reduced in bothdonors.

FIGS. 7A-7B. Cell-free supernatant from distinct neonatal gut microbiomeisolates of Candida consistently induce CD4+ IL4+ (Th2) cells (FIG. 7A).Specific species induce or suppress CD4+ IL10+ (T-reg) cells (FIG. 7B).BHI, sterile brain heart infusion medium exposure (control); CT, C.tropicalis; CP, C. parapsilosis; CO, C. orthopsilosis; and CT, C.tropicalis; Control, non-antigen stimulated conditions; CRA, cockroachstimulated T-cells.

FIGS. 8A-8D. UC microbiotypes exhibit significantly different diseaseseverity and duration. FIG. 8A: Simple colitis disease severity score.FIG. 8B: Number of extra-colonic manifestations. FIG. 8C: Diseaseduration. FIG. 8D. Number of family members with inflammatory boweldisease (IBD).

FIGS. 9A-9D. In vitro fecal water assays reveal that UC patients exhibitsignificantly distinct Th2 ratios, IL4 production and CD8+ IL17+populations. FIG. 9A: A significant skew towards Th2 responsescharacterizes UC patients compared to healthy controls. FIG. 9B: UCmicrobiotypes exhibit significant differences in the degree of Th2 skew,with the most severe microbiotype (MBT-1) exhibiting the most profoundTh2 skew. FIG. 9C: IL4 expression is significantly different across UCmicrobiotypes, with the MBT-1 group exhibiting significantly higher IL4expression compared to the lowest disease severity group. FIG. 9D: MBT-1patients exhibit significantly greater numbers of CD8+ IL17+ cellscompared with the two other lower disease severity groups.

FIG. 10. Experimental timeline illustrating phosphate buffered saline(PBS) or the therapeutic consortium (TC) supplementation regime and CRAchallenge schedule in a murine model of airway allergic sensitization.

FIGS. 11A-11B. Oral supplementation of mice with the TC promotesincreased relative abundance of genera associated with induction ofimmune tolerance. FIG. 11A: Microbiome composition was determined in thefeces of the animals in the study using 16S rRNA sequencing. Clusteranalysis revealed differences in microbiome composition across treatmentgroups. The TC-supplemented animals showed a significantly distinctcomposition compared with the control groups. Specifically,TC-supplemented animals were enriched for species with the potential forimmunomodulatory activity (e.g., Bifidobacterium, Clostridia speciesbelong to Clade IV and XIV, Lachnospira, and Bacteroides). FIG. 11B: Piechart showing enriched taxa in CRA-TC treated animals.

FIGS. 12A-12B. Oral supplementation of mice with the TC promotesmetabolic reprogramming in both the gut lumen and periphery and includessignificant increases in circulating itaconate, which is associated witha repair macrophage effector phenotype. FIG. 12A: Principle componentsanalysis of the dominant luminal metabolites using un-targeted liquidchromatography mass spectrometry revealed distinct metabolic profilesbetween the three groups (Canberra Distance Matrices; PERMANOVA,R²=0.29, p=0.005). FIG. 12B: Principle components analysis of thecirculating metabolites identified in the serum using the same strategyalso revealed significant differences (Canberra Distance Matrices;PERMANOVA, R²=0.29, p=0.002 between the groups examined. Untargeted LCGC Mass spectrometry was used to identify and determine the relativeconcentrations of several hundred metabolites in the feces (FIG. 12A)and serum (FIG. 12B) of mice supplemented with the TC or PBS prior tocockroach (CRA) antigen challenge. Significant spatial separation ofTC-supplemented versus PBS supplemented animals on a PcoA plot indicatesthat the profile of metabolites in the feces and serum of these animalsis significantly different.

FIGS. 13A-13B. FIG. 13A: Histological sections of the murine airway(lung) indicate that oral supplementation of mice with the metabolicallyactive therapeutic consortium (CRA+TC) significantly reducesinflammatory influx [Hemotoxylin and Eosin (H&E) Staining; dark stainednucleated cells] in a murine model of airway allergic sensitization. L.johnsonii alone does not confer protection (CRA+Lj), nor doessupplementation of animals with four of the TC (omitting L. johnsonii;CRA+C), indicating that L. johnsonii acts in synergy with the other fourmembers of the TC to effect protection at the airway mucosal surface. Aheat-killed, metabolically inactive TC also does not confer protectionindicating that only the metabolically active TC protects. FIG. 13B:Gene expression analyses of CCL-11 expression, a marker of eosinophils,confirm that the CRA+TC group exhibits significantly reduced eosinophilpresence in the lungs following allergic sensitization.

FIG. 14A-14B. FIG. 14A: Histological sections of the murine airway(lung) indicate that oral supplementation of mice with the metabolicallyactive therapeutic consortium (CRA+TC) significantly reduces mucinhyper-secretion (Periodic Acid-Schiff (PAS) Staining: dark staining) ina murine model of airway allergic sensitization. L. johnsonii alone doesnot reduce mucin secretion (CRA+Lj), nor does supplementation of animalswith four of the TC (omitting L. johnsonii; CRA+C), indicating that L.johnsonii acts in synergy with the other four members of the TC tosuppress mucin secretion at the airway mucosal surface. A heat-killed,metabolically inactive TC also does not reduce mucin secretionindicating that only the metabolically active TC protects. FIG. 14B:Gene expression analyses of Muc5AC expression, the primary generesponsible for mucin secretion in the airways, confirm that the CRA+TCgroup exhibits significantly reduced mucin gene expression the lungs,compared to the other treatment groups.

FIGS. 15A-15C. Oral supplementation of mice with the metabolicallyactive TC significantly reduces cytokine expression associated withallergic inflammation in a murine model of airway allergicsensitization. FIG. 15A: Boxplot demonstrating a significant decrease inrelative change in expression of IL-13 in animals treated with TC andCRA challenged compared with PBS treatment and CRA challenged. FIG. 15B:Boxplot demonstrating a significant decrease in relative change inexpression of IL-4 in animals treated with TC and CRA challengedcompared with PBS treatment and CRA challenge. FIG. 15C: Boxplotdemonstrating a significant decrease in relative change in expression ofIL-10 in animals treated with TC and CRA challenged compared with PBStreatment and CRA challenge.

FIGS. 16A-16F. Oral supplementation of mice with the TC results in arepair macrophage effector phenotype in a murine model of airwayallergic sensitization. In CRA+TC treated mice, CD11b^(hi)F4/80^(hi)macrophages form a larger percentage of the non-lymphocyte population in(FIG. 16A) mesenteric lymph nodes, (FIG. 16B) spleen, and (FIG. 16C)lung compared to CRA+PBS treated animals. CRA+TC and CRA+PBS treatedanimals show similar percentages of CD11b^(hi)F4/80^(hi) CD206⁺ (M2)macrophages in the non-lymphocyte population in both (FIG. 16D)mesenteric lymph nodes and (FIG. 16E) spleen. FIG. 16F: In lung, CRA+TCtreated animals show an increased percentage of CD11b^(hi)F4/80^(hi)CD206⁺ (M2) macrophages in the non-lymphocyte population compared toCRA+PBS treated mice.

FIG. 17. Table showing treatment groups utilized in murine model ofairway allergic sensitization study.

FIGS. 18A-18C. Metabolic reprogramming in gut lumen following oralsupplementation of mice with the therapeutic consortium (TC) promotesincreased concentrations of specialized lipids, plasmalogens, which areenriched in polyunsaturated fatty acids (PUFAs). PUFAs are increased inthe feces of neonates at low risk for allergies and asthma in childhood.FIG. 18A: Carbohydrate compounds decreased in concentration followingtreatment with the TC. FIG. 18B: Energy compounds decreased inconcentration following treatment with the TC. FIG. 18C: Lipid compoundsdecreased in concentration (PUFAs, Long chain fatty acids,acyl-glycerols, and branched fatty acids) and increased (phospholipidsand plasmalogens) following treatment with the TC.

FIG. 19. Bacterial and fungal α- and β-diversity are related toparticipant age at the time of fecal-sample collection. Bacterial andfungal α-diversities are inversely correlated (Shannon's index; n=188;Pearson's correlation, r²=−0.24; P<0.001).

FIGS. 20A-20B. Compositionally distinct, age-independent NGM statesexist in neonates, exhibit significant differences in fungal taxonomyand are related to the RR of atopy at the age of 2 years. FIG. 20A: NGMparticipants do not differ significantly in age (n=130; Kruskal-Wallis;P=0.256). Box plots are defined by the 25^(th) and 75^(th) percentiles.Center line represents the median (50^(th) percentile). Whiskers aredefined as 1.5 times the interquartile range (IQR, 75^(th)-25^(th)percentile), plus or minus the 75^(th) and 25^(th) percentiles,respectively. FIG. 20B: The sum of allergen-specific serum IgEconcentrations measured at 2 years of age (n=130) is significantlyhigher for NGM3 compared with NGM1 participants (Welch's t test;P=0.034). Box plots are constructed as defined in FIG. 20A.

FIG. 21. NGMs exhibit significantly different RRs of PM atopydevelopment at age 2 years and of parental report of doctor-diagnosedasthma at age 4 years. Significance of risk ratios between microbiotastates was calculated on the basis of log-binomial regression.

FIGS. 22A-22F. Sterile fecal water from NGM3 participants induces CD4⁺cell population dysfunction associated with atopic asthma. Dendriticcells and autologously purified naïve CD4⁺ cells from the serum of twohealthy adult donors (biological replicates) were incubated with sterilefecal water from NGM1 (n=7; three biological replicates per sample) orNGM3 (n=5; three biological replicates per sample) participants. FIGS.22A and 22B: Fecal water from NGM3 participants induced significantlyincreased proportions of CD4⁺IL-4⁺ cells (LME, P<0.001; center linerepresents mean) (FIG. 22A) and expression of IL-4 (LME; P=0.045) (FIG.22B). FIG. 22C: Fecal water from both NGM1 and NGM3 participants inducedsignificantly increased proportions of CD4⁺CD25⁺FOXP3⁺ cells (LME;P<0.001 for NGM1 and P=0.017 for NGM3), compared with control. FIG. 22D:Weighted correlation network analysis identified a metabolic module thatdifferentiates NGM3 from NGM2 and NGM1 participants (n=28; ANOVA;P=0.038). Box plots define the 25^(th) and 75^(th) percentiles; themedian is represented by the center line. IQR (75^(th)-25^(th)percentile) is represented by whiskers. FIG. 22E: Scatterplot ofmetabolite significance versus module membership (MM) of the 12metabolites in the NGM3-discriminating metabolic module. Metaboliteswith a higher metabolite significance value discriminate NGM3 from otherNGMs. Metabolites plotted above the dashed line (representing theoverall p-value for between-NGM differences) are significantlyassociated with NGM differentiation (P<0.05), and were detected inhigher concentrations in NGM3 compared to the other NGMs. MM valuesindicate the degree of interconnectedness of a specific metabolite toother metabolites in the module (higher MM value indicates greaterinterconnectedness). FIG. 22F: When the same ex vivo assay that wasperformed in FIGS. 22A-22C was used, 12,13-DiHOME significantly reducedthe proportion of CD4⁺CD25⁺FOXP3⁺ cells at three differentconcentrations compared to vehicle control (LME; P=0.04, P<0.001,P=0.001 for concentrations of 75, 130 and 200 μM, respectively; centerline represents mean proportion of cells).

FIG. 23. Dirichlet multinomial mixture model identifies threecompositionally distinct bacterial NGMs as the best model fit. Model fitwas based on the Laplace approximation to the negative log model where alower value indicates a better model fit.

FIG. 24. Sterile fecal water from NGM3 participants induces a CD4⁺IL-4⁺cell skew. Dendritic cells from serum of two healthy adult donors(biological replicates), were incubated with sterile fecal water fromNGM1 (n=7; three biological replicates per sample) or NGM3 (n=5; threebiological replicates per sample) participants, prior to co-incubationwith autologously purified naïve CD4⁺ cells. NGM3 fecal water induces atrend toward a CD4⁺IL-4⁺ cell skew compared with NGM1 (LME; P=0.095).

FIG. 25. Confirmation that the concentration of the dihydroxy fatty acid12, 13 DiHOME, is significantly increased in the NGM3 sample subset usedfor ex vivo assays. Using the subset of samples employed in the ex vivoDC-T-cell assay and based on metabolite scaled intensity data, 12, 13DiHOME is significantly increased in relative concentration in NGM3(n=7) compared to NGM1 (n=5) samples (Welch's-test; P=0.033).

FIG. 26. Allergens used to determine PM atopy status of participants inthis study. Mean and median of allergen-specific IgE (IU ml⁻¹) isprovided for each.

FIG. 27. Risk ratio of IGMs (infants>6 months old) for developing atopyor having parental report of doctor's diagnosis of asthma. Risk ratioswere calculated based on log-binomial regression.

FIG. 28. Fungal taxa exhibiting significantly increased relativeabundance in lower-risk NGM1 versus higher-risk NGM3 neonatal gutmicrobiota. Significant difference in relative abundance was determinedusing a zero-inflated negative binomial regression model (q<0.20). Whitebackground indicates taxa enriched in NGM1 (compared with NGM3), graybackground indicates taxa enriched in NGM3 (compared with NGM1).Findings are ranked by difference in relative abundance (NGM1-NGM3).

FIG. 29. Fungal taxa exhibiting significantly increased relativeabundance in lower-risk NGM2 versus higher-risk NGM3 neonatal gutmicrobiota. Significant difference in relative abundance was determinedusing zero-inflated negative binomial regression model (q<0.20). Whitebackground indicates taxa enriched in NGM2 (compared with NGM3), graybackground indicates taxa enriched in NGM3 (compared with NGM2).Findings are ranked by difference in relative abundance (NGM2-NGM3).

FIG. 30. Procrustes analyses of 16S rRNA-based β-diversity, PICRUSt andmetabolomic datasets. Results from Procrustes analyses indicate thatbacterial β-diversity, PICRUSt and metabolomic data is highly andsignificantly correlated.

FIGS. 31A-31C. Comparison of healthy (n=13) and UC-associated (n=30)fecal microbiotas. FIG. 31A: Bacterial diversity. Horizontal barsrepresent means±standard deviations. P values were obtained bytwo-tailed Student t test. FIG. 31B: Bacterial community compositionrepresented by nonmetric multidimensional scaling (NMDS) of pairwiseweighted UniFrac distances. FIG. 31C: Bacterial community composition ofUC patients stratified by ethnicity (18 EU UC, 12 SA UC) represented byNMDS of pairwise weighted UniFrac distances. In FIG. 31B and FIG. 31C,each dashed ellipse represents the 95% confidence interval for thecentroid of each stratification group as calculated by ordiellipse.

FIGS. 32A-32D. Clinical measurements of UC severity among UC MCSs (11for MCS1, 8 for MCS2, 4 for MCS3, 3 for MCS4). FIG. 32A: Simple clinicalcolitis activity. FIG. 32B: Number of extracolonic symptoms. FIG. 32C:Number of family members diagnosed with IBD. FIG. 32D: Duration ofdisease. All pairwise comparisons were done with a two-tailed Dunn test.Only P values of <0.1 are indicated. EU UC, squares; SA UC, circles.

FIG. 33A-33K. In vitro human T-cell activity following coculture withautologous DCs coincubated with sterile fecal water. FIG. 33A:Th1-to-Th2 ratio; FIG. 33B: Th1 frequency; FIG. 33C: Th2 frequency; FIG.33D: Th17 frequency; e, regulatory T-cell frequency (48 healthy, 116UC). Comparisons of the Th1 frequencies (FIG. 33F), Th2 frequencies(FIG. 33G), and Th1-to-Th2 ratios (FIG. 33H) of healthy and UC MCSs areshown (48 for healthy, 48 for MCS1, 40 for MCS2, 16 for MCS3, and 8 forMCS4). Concentrations of IL-4 (FIG. 33I), IL-5 (FIG. 33J), and IL-13(FIG. 33K) in cell supernatant following coculture of human T cells withautologous DCs challenged with sterilized fecal water from healthyparticipants and MCS1 and MCS2 patients are shown (48 for healthyparticipants, 48 for MCS1 patients, and 40 for MCS2 patients). Data weregenerated from four (FIGS. 33A-33H) or two (FIG. 33I-33K) replicateexperiments with DCs/T cells obtained from two anonymous PBMC donors.Horizontal bars (mean fitted values for each group) and P values weredetermined by linear mixed-effect modeling (see Materials and Methods).P values of <0.1 are indicated.

FIGS. 34A-34H. Comparison of healthy (n=13) and UC-associated (n=30)fecal fungal microbiotas. FIG. 34A: Fungal α diversity stratified byhealthy status. FIG. 34B: Fungal community composition represented byNMDS of pairwise Bray-Curtis distances. Participants are colored byhealth status. Bacterial α diversity FIG. 34C and fungal α diversityFIG. 34D were stratified by health status and ethnicity (10 healthy EU,3 healthy SA, 18 UC EU, 12 UC SA). FIG. 34E: Simple clinical colitisactivity of UC patients stratified by ethnicity (14 EU UC, 12 SA UC). Pvalues were obtained by two-tailed rank sum test. FIG. 34F Bacterialcommunity composition of all participants stratified by ethnicity (28EU, 15 SA) represented by NMDS of pairwise weighted UniFrac distances.FIG. 34G: Fungal community composition of all participants stratified byethnicity (28 EU, 15 SA) represented by NMDS of pairwise Bray-Curtisdistances. FIG. 34H: PhyloChip-profiled bacterial community compositionof UC patients stratified by ethnicity (15 EU UC, 11 SA UC) representedby NMDS of pairwise Canberra distances. In panels a, c, and d,horizontal bars represent means±standard deviations. P values wereobtained by two-tailed t test. In FIG. 34B and FIGS. 34F-34H, eachdashed ellipse represents the 95% confidence interval for the centroidof each participant stratification group as calculated by ordiellipse.Each dot/square represents a single fecal sample obtained from a singledonor.

FIGS. 35A-35B. Bacterial community compositions of UC patientsstratified by UC MCS. FIG. 35A: NMDS of pairwise weighted UniFracdistances for 16S rRNA profiles obtained via Illumina MiSeq (12 MCS1, 10MCS2, 4 MCS3, 3 MCS4, 1 other). FIG. 35B: NMDS of pairwise Canberradistances for 16S rRNA profiles obtained via PhyloChip (10 MCS1, 8 MCS2,4 MCS3, 2 MCS4, 1 other). Each dashed ellipse represents the 95%confidence interval for the centroid of each participant stratificationgroup as calculated by ordiellipse. Each dot/square represents a singlefecal sample obtained from a single donor.

FIG. 36. In vitro human T-cell activity following coculture withautologous DCs coincubated with sterile fecal water. Induced Th1-to-Th2ratios of EU UC (n=) and SA UC patients are compared. Data weregenerated from four replicate experiments with DCs/T cells obtained fromtwo anonymous PBMC donors. Horizontal bars (mean fitted values for eachgroup) and P values were determined by linear mixed-effect modeling.

FIG. 37. Breakdown of Study Participant Cohort. Note: one SA-UCparticipant failed to report their sex.

FIG. 38. Description of Metabolon QC Samples.

FIG. 39. Metabolon QC Standards.

DETAILED DESCRIPTION I. Definitions

While various embodiments and aspects of the present invention are shownand described herein, it will be obvious to those skilled in the artthat such embodiments and aspects are provided by way of example only.Numerous variations, changes, and substitutions will now occur to thoseskilled in the art without departing from the invention. It should beunderstood that various alternatives to the embodiments of the inventiondescribed herein may be employed in practicing the invention.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in the applicationincluding, without limitation, patents, patent applications, articles,books, manuals, and treatises are hereby expressly incorporated byreference in their entirety for any purpose.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York,N.Y. 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL,Cold Springs Harbor Press (Cold Springs Harbor, N Y 1989). Any methods,devices and materials similar or equivalent to those described hereincan be used in the practice of this invention. The following definitionsare provided to facilitate understanding of certain terms usedfrequently herein and are not meant to limit the scope of the presentdisclosure.

The term “exogenous” refers to a molecule or substance (e.g., acompound, nucleic acid or protein) that originates from outside a givencell or organism. For example, an “exogenous promoter” as referred toherein is a promoter that does not originate from the plant it isexpressed by. Conversely, the term “endogenous” or “endogenous promoter”refers to a molecule or substance that is native to, or originateswithin, a given cell or organism.

The term “isolated”, when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It can be,for example, in a homogeneous state and may be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified.

The term “isolated”, when applied to a bacterium, refers to a bacteriumthat has been (1) separated from at least some of the components withwhich it was associated when initially produced (whether in nature or inan experimental setting), and/or (2) produced, prepared, purified,and/or manufactured by the hand of man, e.g. using artificial cultureconditions such as (but not limited to) culturing on a plate and/or in afermenter. Isolated bacteria include those bacteria that are cultured,even if such cultures are not monocultures. Isolated bacteria may beseparated from at least about 10%, about 20%, about 30%, about 40%,about 50%, about 60%, about 70%, about 80%, about 90%, or more of theother components with which they were initially associated. Inembodiments, isolated bacteria are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%6, or more than about 99% pure. Inembodiments, a bacterial population provided herein comprises isolatedbacteria. In embodiments, a composition provided herein comprisesisolated bacteria. In embodiments, the bacteria that are administeredare isolated bacteria.

As used herein, a substance is “pure” if it is substantially free ofother components. The terms “purify,” “purifying” and “purified”, whenapplied to a bacterium, refer to a bacterium that has been separatedfrom at least some of the components with which it was associated eitherwhen initially produced or generated (e.g., whether in nature or in anexperimental setting), or during any time after its initial production.A bacterium or a bacterial population may be considered purified if itis isolated at or after production, such as from a material orenvironment containing the bacterium or bacterial population, or bypassage through culture, and a purified bacterium or bacterialpopulation may contain other materials up to about 10%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,or above about 90% and still be considered “isolated.” In someembodiments, purified bacteria and bacterial populations are more thanabout 80%, about 85%, about 90%0, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more thanabout 99% pure. In the instance of microbial compositions providedherein, the one or more bacterial types (species or strains) present inthe composition can be independently purified from one or more otherbacteria produced and/or present in the material or environmentcontaining the bacterial type. Microbial compositions and the bacterialcomponents thereof are generally purified from residual habitatproducts.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues,wherein the polymer may In embodiments be conjugated to a moiety thatdoes not consist of amino acids. The terms apply to amino acid polymersin which one or more amino acid residue is an artificial chemicalmimetic of a corresponding naturally occurring amino acid, as well as tonaturally occurring amino acid polymers and non-naturally occurringamino acid polymers. A “fusion protein” refers to a chimeric proteinencoding two or more separate protein sequences that are recombinantlyexpressed as a single moiety.

The term “peptidyl” and “peptidyl moiety” means a monovalent peptide.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. The terms“non-naturally occurring amino acid” and “unnatural amino acid” refer toamino acid analogs, synthetic amino acids, and amino acid mimetics whichare not found in nature.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences. Because of the degeneracy of the genetic code, a number ofnucleic acid sequences will encode any given protein. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide is implicit ineach described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins (1984)).

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over aspecified region, when compared and aligned for maximum correspondenceover a comparison window or designated region) as measured using a BLASTor BLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like).Such sequences are then said to be “substantially identical.” Thisdefinition also refers to, or may be applied to, the compliment of atest sequence. The definition also includes sequences that havedeletions and/or additions, as well as those that have substitutions. Asdescribed below, the preferred algorithms can account for gaps and thelike. Preferably, identity exists over a region that is at least about25 amino acids or nucleotides in length, or more preferably over aregion that is 50-100 amino acids or nucleotides in length.

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include 32P,fluorescent dyes (e.g. cyanine), electron-dense reagents, enzymes (e.g.,as commonly used in an ELISA), biotin, digoxigenin, or haptens andproteins or other entities which can be made detectable, e.g., byincorporating a radiolabel into a peptide or antibody specificallyreactive with a target peptide. Any appropriate method known in the artfor conjugating an antibody to the label may be employed, e.g., usingmethods described in Hermanson, Bioconjugate Techniques 1996, AcademicPress, Inc., San Diego.

“Contacting” is used in accordance with its plain ordinary meaning andrefers to the process of allowing at least two distinct species (e.g.chemical compounds including biomolecules and/or cells such as bacterialcells) to become sufficiently proximal to react, interact or physicallytouch. It should be appreciated; however, a resulting reaction productcan be produced directly from a reaction between the added reagents orfrom an intermediate from one or more of the added reagents which can beproduced in the reaction mixture.

The term “contacting” may include allowing two species to react,interact, or physically touch, wherein the two species may be, forexample, an antibody domain as described herein and an antibody-bindingdomain. In embodiments contacting includes, for example, allowing anantibody domain as described herein to interact with an antibody-bindingdomain.

“Patient” or “subject in need thereof” refers to a living member of theanimal kingdom suffering from or that may suffer from the indicateddisorder. In embodiments, the subject is a member of a speciescomprising individuals who naturally suffer from the disease. Inembodiments, the subject is a mammal. Non-limiting examples of mammalsinclude rodents (e.g., mice and rats), primates (e.g., lemurs,bushbabies, monkeys, apes, and humans), rabbits, dogs (e.g., companiondogs, service dogs, or work dogs such as police dogs, military dogs,race dogs, or show dogs), horses (such as race horses and work horses),cats (e.g., domesticated cats), livestock (such as pigs, bovines,donkeys, mules, bison, goats, camels, and sheep), and deer. Inembodiments, the subject is a human. In embodiments, the subject is anon-mammalian animal such as a turkey, a duck, or a chicken. Inembodiments, a subject is a living organism suffering from or prone to adisease or condition that can be treated by administration of acomposition or pharmaceutical composition as provided herein.

The terms “disease” or “condition” refer to a state of being or healthstatus of a patient or subject capable of being treated with a compound,pharmaceutical composition, or method provided herein. In embodiments,the disease is an inflammatory disease (e.g. asthma, ulcerative colitis,irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum,erythema nodosum, or any other inflammatory disease mentioned herein).As used herein, a “symptom” of a disease includes any clinical orlaboratory manifestation associated with the disease, and is not limitedto what a subject can feel or observe.

As used herein, the term “inflammatory disease” refers to a disease orcondition characterized by aberrant inflammation (e.g., an increasedlevel of inflammation compared to a control such as a healthy person notsuffering from a disease). Non-limiting examples of inflammatorydiseases include allergy, atopy, asthma, an autoimmune disease, anautoinflammatory disease, a hypersensitivity, pediatric allergic asthma,allergic asthma, inflammatory bowel disease, Celiac disease, Crohn'sdisease, colitis, ulcerative colitis, collagenous colitis, lymphocyticcolitis, diverticulitis, irritable bowel syndrome, short bowel syndrome,stagnant loop syndrome, chronic persistent diarrhea, intractablediarrhea of infancy, Traveler's diarrhea, immunoproliferative smallintestinal disease, chronic prostatitis, postenteritis syndrome,tropical sprue, Whipple's disease, Wolman disease, arthritis, rheumatoidarthritis, Behçet's disease, uveitis, pyoderma gangrenosum, erythemanodosum, traumatic brain injury, psoriatic arthritis, juvenileidiopathic arthritis, multiple sclerosis, systemic lupus erythematosus(SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitustype 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto'sthyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome,vasculitis, glomerulonephritis, auto-immune thyroiditis, bullouspemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison'sdisease, Vitiligo, acne vulgaris, pelvic inflammatory disease,reperfusion injury, sarcoidosis, transplant rejection, interstitialcystitis, atherosclerosis, and atopic dermatitis.

As used herein the term “dysbiosis” means a difference in thegastrointestinal microbiota compared to a healthy or general population.In embodiments, dysbiosis comprises a difference in gastrointestinalmicrobiota commensal species diversity compared to a healthy or generalpopulation. In an embodiment, dysbiosis comprises a decrease ofbeneficial microorganisms and/or increase of pathobionts (pathogenic orpotentially pathogenic microorganisms) and/or decrease of overallmicrobiota species diversity. Many factors can harm the beneficialmembers of the intestinal microbiota leading to dysbiosis, including(but not limited to) antibiotic use, psychological and physical stress,radiation, and dietary changes. In an embodiment, dysbiosis comprises orpromotes the overgrowth of a bacterial opportunistic pathogen such asEnterococcus faecalis, Enterococcus faecium, or Clostridium difficile.In an embodiment, the dysbiosis comprises a reduced amount (absolutenumber or proportion of the total microbial population) of bacterial orfungal cells of a species or genus (e.g., 5%, 10%, 15%, 20%¹, 25%, 30%,35%, 40%, 45%/O, 50%, 55%, 600, 65%, 70%, 75%, 80%, 85%, 90%, 95% ormore lower) compared to a healthy subject (e.g., a corresponding subjectwho does not have an inflammatory disease, an infection, and who has notbeen administered an antibiotic within about 1, 2, 3, 4, 5, or 6 months,and/or compared to a healthy or general population). In an embodiment,the dysbiosis comprises an increased amount (absolute number orproportion of the total microbial population) of bacterial or fungalcells within a species or genus (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%/O or morehigher) compared to a healthy subject (e.g., a corresponding subject whodoes not have an inflammatory disease, an infection, and who has notbeen administered an antibiotic within about 1, 2, 3, 4, 5, or 6 months,and/or compared to a healthy or general population). In an embodiment, asubject who comprises a gastrointestinal infection, gastrointestinalinflammation, diarrhea, colitis, or who has received an antibioticwithin about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks is deemed tocomprise dysbiosis. In an embodiment, the impaired microbiota comprisessmall intestinal bacterial or fungal overgrowth. Antibioticadministration (e.g., systemically, such as by intravenous injection ororally) is a common and significant cause of major alterations in thenormal microbiota. Thus, as used herein, the term “antibiotic-induceddysbiosis” refers to dysbiosis caused by or following the administrationof an antibiotic.

Non-limiting examples of dysbiosis are described in the examplesprovided herein. Non-limiting examples of dysbiosis in the context ofneonates are also described in Fujimura et al. (2016) “Neonatal gutmicrobiota associates with childhood multisensitized atopy and T celldifferentiation” Nature Medicine 22(10): 1187-1191 (hereinafter“Fujimura et al. 2016”), the entire content of which (including allsupplemental information and data) is incorporated herein by reference.In some embodiments, a subject with dysbiosis has the NGM3 microbiomeprofile as set forth in Fujimura et al. 2016. Non-limiting examples ofdysbiosis in the context of ulcerative colitis are described in Mar etal. (2016) “Disease Severity and Immune Activity Relate to DistinctInterkingdom Gut Microbiome States in Ethnically Distinct UlcerativeColitis Patients” mBio 7(4):e01072-16 (herein after “Mar et al. 2016”),the entire content of which (including all supplemental information anddata) is incorporated herein by reference. In some embodiments, asubject with dysbiosis has the MCS4 microbiome profile as set forth inMar et al. 2016. In some embodiments, a subject with dysbiosis has theMCS 3 microbiome profile as set forth in Mar et al. 2016. In someembodiments, a subject with dysbiosis has the MCS2 microbiome profile asset forth in Mar et al. 2016. In some embodiments, a subject withdysbiosis has the MCS1 microbiome profile as set forth in Mar et al.2016.

The term “associated” or “associated with” in the context of a substanceor substance activity or function associated with a disease (e.g., anallergy, asthma, ulcerative colitis, irritable bowel syndrome,arthritis, uveitis, pyoderma gangrenosum, or erythema nodosum) meansthat the disease is caused by (in whole or in part), or a symptom of thedisease is caused by (in whole or in part) the substance or substanceactivity or function.

The term “aberrant” as used herein refers to different from normal. Whenused to describe enzymatic activity, aberrant refers to activity that isgreater or less than a normal control or the average of normalnon-diseased control samples. Aberrant activity may refer to an amountof activity that results in a disease, wherein returning the aberrantactivity to a normal or non-disease-associated amount (e.g. by using amethod as described herein), results in reduction of the disease or oneor more disease symptoms.

A “control” or “standard control” refers to a sample, measurement, orvalue that serves as a reference, usually a known reference, forcomparison to a test sample, measurement, or value. For example, a testsample can be taken from a patient suspected of having a given disease(e.g. dysbiosis, an autoimmune disease, inflammatory autoimmune disease,cancer, infectious disease, immune disease, or other disease) andcompared to a known normal (non-diseased) individual (e.g. a standardcontrol subject). A standard control can also represent an averagemeasurement or value gathered from a population of similar individuals(e.g. standard control subjects) that do not have a given disease (i.e.standard control population), e.g., healthy individuals with a similarmedical background, same age, weight, etc. A standard control value canalso be obtained from the same individual, e.g. from an earlier-obtainedsample from the patient prior to disease onset. For example, a controlcan be devised to compare therapeutic benefit based on pharmacologicaldata (e.g., half-life) or therapeutic measures (e.g., comparison of sideeffects). Controls are also valuable for determining the significance ofdata. For example, if values for a given parameter are widely variant incontrols, variation in test samples will not be considered assignificant. One of skill will recognize that standard controls can bedesigned for assessment of any number of parameters (e.g. microbiome,RNA levels, protein levels, specific cell types, specific bodily fluids,specific tissues, synoviocytes, synovial fluid, synovial tissue,fibroblast-like synoviocytes, macrophage-like synoviocytes, etc).

One of skill in the art will understand which standard controls are mostappropriate in a given situation and be able to analyze data based oncomparisons to standard control values. Standard controls are alsovaluable for determining the significance (e.g. statisticalsignificance) of data. For example, if values for a given parameter arewidely variant in standard controls, variation in test samples will notbe considered as significant.

The term “diagnosis” refers to a relative probability that a disease(e.g. an autoimmune, inflammatory autoimmune, cancer, infectious,immune, or other disease) is present in the subject. Similarly, the term“prognosis” refers to a relative probability that a certain futureoutcome may occur in the subject with respect to a disease state. Forexample, in the context of the present invention, prognosis can refer tothe likelihood that an individual will develop a disease (e.g. anautoimmune, inflammatory autoimmune, cancer, infectious, immune, orother disease), or the likely severity of the disease (e.g., duration ofdisease). The terms are not intended to be absolute, as will beappreciated by any one of skill in the field of medical diagnostics.

“Biological sample” or “sample” refer to materials obtained from orderived from a subject or patient. A biological sample includes sectionsof tissues such as biopsy and autopsy samples, and frozen sections takenfor histological purposes. Such samples include bodily fluids such asblood and blood fractions or products (e.g., serum, plasma, platelets,red blood cells, and the like), feces and feces fractions or products(e.g., fecal water, such as but not limited to fecal water separatedfrom other fecal components and solids by methods such as centrifugationand filtration) sputum, tissue, cultured cells (e.g., primary cultures,explants, and transformed cells), stool, urine, synovial fluid, jointtissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes,macrophage-like synoviocytes, immune cells, hematopoietic cells,fibroblasts, macrophages, dendritic cells, T-cells, etc. In embodiments,a sample is obtained from a eukaryotic organism, such as a mammal suchas a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g.,guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.

A “cell” as used herein, refers to a cell carrying out metabolic orother functions sufficient to preserve or replicate its genomic DNA. Acell can be identified by well-known methods in the art including, forexample, presence of an intact membrane, staining by a particular dye,ability to produce progeny or, in the case of a gamete, ability tocombine with a second gamete to produce a viable offspring. Cells mayinclude prokaryotic and eukaroytic cells. Prokaryotic cells include butare not limited to bacteria. Eukaryotic cells include but are notlimited to yeast cells and cells derived from plants and animals, forexample mammalian, insect (e.g., spodoptera) and human cells. Cells maybe useful when they are naturally nonadherent or have been treated notto adhere to surfaces, for example by trypsinization.

As used herein the abbreviation “sp.” for species means at least onespecies (e.g., 1, 2, 3, 4, 5, or more species) of the indicated genus.The abbreviation “spp.” for species means 2 or more species (e.g., 2, 3,4, 5, 6, 7, 8, 9, 10 or more) of the indicated genus. In embodiments,methods and compositions provided herein comprise a single specieswithin an indicated genus or indicated genera, or 2 or more (e.g., aplurality comprising more than 2) species within an indicated genus orindicated genera. In embodiments, 1, 2, 3, 4, 5, or more or all or theindicated species is or are isolated. In embodiments, the indicatedspecies are administered together. In embodiments, each of the indicatedspecies is present in a single composition that comprises each of thespecies. In embodiments, each of the species is administeredconcurrently, e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 30, or60, 1-5, 1-10, 1-30, 1-60, or 5-15 seconds or minutes of each other.

In this disclosure, “comprises,” “comprising,” “containing,” and“having” and the like can have the meaning ascribed to them in U.S.Patent law and can mean “includes,” “including,” and the like.“Consisting essentially of” or “consists essentially” likewise has themeaning ascribed in U.S. Patent law and the term is open-ended, allowingfor the presence of more than that which is recited so long as basic ornovel characteristics of that which is recited is not changed by thepresence of more than that which is recited, but excludes prior artembodiments. By contrast, the transitional phrase “consisting of”excludes any element, step, or ingredient not specified.

As used herein, the term “about” in the context of a numerical value orrange means±10% of the numerical value or range recited or claimed,unless the context requires a more limited range.

In the descriptions herein and in the claims, phrases such as “at leastone of” or “one or more of” may occur followed by a conjunctive list ofelements or features. The term “and/or” may also occur in a list of twoor more elements or features. Unless otherwise implicitly or explicitlycontradicted by the context in which it is used, such a phrase isintended to mean any of the listed elements or features individually orany of the recited elements or features in combination with any of theother recited elements or features. For example, the phrases “at leastone of A and B;” “one or more of A and B;” and “A and/or B” are eachintended to mean “A alone, B alone, or A and B together.” A similarinterpretation is also intended for lists including three or more items.For example, the phrases “at least one of A, B, and C;” “one or more ofA, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, Balone, C alone, A and B together, A and C together, B and C together, orA and B and C together.” In addition, use of the term “based on,” aboveand in the claims is intended to mean, “based at least in part on,” suchthat an unrecited feature or element is also permissible.

It is understood that where a parameter range is provided, all integerswithin that range, and tenths thereof, are also provided by theinvention. For example, “0.2-5 mg” is a disclosure of 0.2 mg, 0.3 mg,0.4 mg, 0.5 mg, 0.6 mg etc. up to and including 5.0 mg.

As used in the description herein and throughout the claims that follow,the meaning of “a,” “an,” and “the” includes plural reference unless thecontext clearly dictates otherwise.

II. Bacterial Populations and Microbial Compositions

In an aspect, a composition comprising a bacterial population thatcomprises, consists essentially of, or consists of, 1, 2, 3, 4, 5, 6, 7,or 8 (or at least 1, 2, 3, 4, 5, 6, 7, or 8) bacterial species. Inembodiments, the bacterial population comprises, consists essentiallyof, or consists of any 1, 2, 3, 4, 5, 6, 7, or 8 of Lactobacillus sp.,Faecalibacterium sp., Akkermansia sp., Myxococcus sp., Cystobacter sp.,Pediococcus sp., Bifidobacterium sp., and Clostridium sp. Inembodiments, the bacterial population comprises Lactobacillus sp. andFaecalibacterium prausnitzii. In embodiments, the bacterial populationcomprises Lactobacillus sp. and Akkermansia muciniphila. In embodiments,the bacterial population comprises Lactobacillus sp., and Myxococcusxanthus. In embodiments, the bacterial population comprisesLactobacillus sp. and Cystobacter fuscus. In embodiments, the bacterialpopulation comprises Lactobacillus sp. and Pediococcus pentosaceus,Pediococcus acidilactici, Pediococcus damnosus, Pediococcusethanolidurans, or Pediococcus parvulus. In embodiments, the bacterialpopulation comprises Lactobacillus sp. and Bifidobacterium bifidum,Bifidobacterium pseudolongum, Bifidobacterium saeculare, orBifidobacterium subtile. In embodiments, the bacterial populationcomprises Lactobacillus sp. and Clostridium hiranonis. In embodiments,the Lactobacillus sp. is Lactobacillus johnsonii, Lactobacillusrhamnosus, Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillusacidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillusbrevis, Lactobacillus coleohominis, Lactobacillus crispatus,Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillusdiolivorans, Lactobacillus farraginis, Lactobacillus fermentum,Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillushelveticus, Lactobacillus hilgardii, Lactobacillus intestinalis,Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacilluskefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillusmanihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillusoligofermentans, Lactobacillus panis, Lactobacillus pantheris,Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillusparakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus,Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae,Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillussucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis. Inembodiments, the Lactobacillus sp. is Lactobacillus johnsonii. Inembodiments, the bacterial population comprises at least 1, 2, 3, 4, 5,6, 7, 8, 9, or 10, or from 1-5, 1-10, 1-5, or 1-20 of any combination ofthe following: Lactobacillus johnsonii, Lactobacillus rhamnosus,Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillusacidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillusbrevis, Lactobacillus coleohominis, Lactobacillus crispatus,Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillusdiolivorans, Lactobacillus farraginis, Lactobacillus fermentum,Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillushelveticus, Lactobacillus hilgardii, Lactobacillus intestinalis,Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacilluskefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillusmanihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillusoligofermentans, Lactobacillus panis, Lactobacillus pantheris,Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillusparakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus,Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae,Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillussucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus vini, Lactococcus garvieae, Lactococcus lactis,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus, Cystobacter fuscus, Pediococcus pentosaceus, Pediococcusacidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, andPediococcus parvulus. In embodiments, the bacterial population includesLactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansiamuciniphila, Myxococcus xanthus, and/or Pediococcus pentosaceus. Inembodiments, the bacteria are isolated bacteria.

In an aspect, a composition including Lactobacillus sp.,Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/orPediococcus sp is provided. In embodiments, (i) the Lactobacillus sp. isLactobacillus johnsonii; (ii) the Faecalibacterium sp., isFaecalibacterium prausnitzii; (iii) the Akkermansia sp. is Akkermansiamuciniphila; (iv) the Myxococcus sp. is Myxococcus xanthus; and (v) thePediococcus sp. is Pediococcus pentosaceus.

In an aspect, a composition including Lactobacillus sp.,Faecalibacterium sp., Akkermansia sp., Cystobacter sp., and Pediococcussp is provided. In embodiments, (i) the Lactobacillus sp. isLactobacillus johnsonii; (ii) the Faecalibacterium sp., isFaecalibacterium prausnitzii; (iii) the Akkermansia sp. is Akkermansiamuciniphila; (iv) the Cystobacter sp. is Cystobacter fuscus; and (v) thePediococcus sp. is Pediococcus pentosaceus.

In embodiments, the bacterial population further comprisesBifidobacterium sp. or Clostridium sp. In embodiments, theBifidobacterium sp. is Bifidobacterium bifidum, Bifidobacteriumpseudolongum, Bifidobacterium saeculare, or Bifidobacterium subtile. Inembodiments, the Clostridium sp. is Clostridium hiranonis.

In an aspect, a microbial composition is provided. The compositionincludes Lactobacillus johnsonii, Faecalibacterium prausnitzii,Akkermansia muciniphila, Myxococcus xanthus, Pediococcus pentosaceus anda biological carrier suitable for administration to the gut.

In an aspect, a microbial composition is provided. The compositionincludes Lactobacillus johnsonii, Faecalibacterium prausnitzii,Akkermansia muciniphila, Myxococcus xanthus or Pediococcus pentosaceusand a biological carrier suitable for administration to the gut.

In embodiments, the biological carrier is suitable for oral or rectaladministration. In embodiments, the biological carrier is suitable forcolonization of the gut. A “biologically acceptable” (or“pharmacologically acceptable”) carrier as referred to herein refers tomolecular entities and compositions as described herein that do notproduce an adverse, allergic or other untoward reaction whenadministered to an animal or a human.

In embodiments, the composition includes less than about 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 different speciesof bacteria. In embodiments, the composition includes less than about 20different species of bacteria. In embodiments, the composition includesless than 20 different species of bacteria. In embodiments, thecomposition includes less than about 15 different species of bacteria.In embodiments, the composition includes less than 15 different speciesof bacteria. In embodiments, the composition includes less than about 10different species of bacteria. In embodiments, the composition includesless than 10 different species of bacteria. In embodiments, thecomposition includes less than about 9 different species of bacteria. Inembodiments, the composition includes less than 9 different species ofbacteria. In embodiments, the composition includes less than about 8different species of bacteria. In embodiments, the composition includesless than 8 different species of bacteria. In embodiments, thecomposition includes less than about 7 different species of bacteria. Inembodiments, the composition includes less than 7 different species ofbacteria. In embodiments, the composition includes less than about 6different species of bacteria. In embodiments, the composition includesless than 6 different species of bacteria. In embodiments, thecomposition includes less than about 5 different species of bacteria. Inembodiments, the composition includes less than 5 different species ofbacteria. In embodiments, the composition includes less than about 4different species of bacteria. In embodiments, the composition includesless than 4 different species of bacteria. In embodiments, thecomposition includes less than about 3 different species of bacteria. Inembodiments, the composition includes less than 3 different species ofbacteria. In embodiments, the composition includes less than about 2different species of bacteria. In embodiments, the composition includesless than 2 different species of bacteria.

In embodiments, the composition is not a fecal transplant. Inembodiments, the composition further includes a pharmaceuticallyacceptable excipient. In embodiments, the composition is a capsule, atablet, a suspension, a suppository, a powder, a cream, an oil, anoil-in-water emulsion, a water-in-oil emulsion, or an aqueous solution.In embodiments, the composition is in the form of a powder, a solid, asemi-solid, or a liquid. In embodiments, the composition is a food or abeverage.

In embodiments, the Lactobacillus sp., the Faecalibacterium sp., theAkkermansia sp., the Myxococcus sp., and/or the Pediococcus sp. is inthe form of a powder. In embodiments, the Lactobacillus sp., theFaecalibacterium sp., the Akkermansia sp., the Myxococcus sp., and/orthe Pediococcus sp. has been lyophilized.

In embodiments, the Myxococcus sp. is in the form of spores, vegetativebacteria, or a mixture of spores and vegetative bacteria. Inembodiments, the Myxococcus sp. is in the form of a powder comprisingspores. In embodiments, the Clostridium sp. is in the form of spores,vegetative bacteria, or a mixture of spores and vegetative bacteria. Inembodiments, the Clostridium sp. is in the form of a powder comprisingspores.

In embodiments, the bacterial composition has a water activity (a_(w))less than about 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 at 20° C.In embodiments, the bacterial composition has an a_(w) less than about0.9 at 20° C. In embodiments, the bacterial composition has an a_(w)less than 0.9 at 20° C. In embodiments, the bacterial composition has ana_(w) less than about 0.8 at 20° C. In embodiments, the bacterialcomposition has an a_(w) less than 0.8 at 20° C. In embodiments, thebacterial composition has an a_(w) less than about 0.7 at 20° C. Inembodiments, the bacterial composition has an a_(w) less than 0.7 at 20°C. In embodiments, the bacterial composition has an a_(w) less thanabout 0.6 at 20° C. In embodiments, the bacterial composition has ana_(w) less than 0.6 at 20° C. In embodiments, the bacterial compositionhas an a_(w) less than about 0.5 at 20° C. In embodiments, the bacterialcomposition has an a_(w) less than 0.5 at 20° C. In embodiments, thebacterial composition has an a_(w) less than about 0.4 at 20° C. Inembodiments, the bacterial composition has an a_(w) less than 0.4 at 20°C. In embodiments, the bacterial composition has an a_(w) less thanabout 0.3 at 20° C. In embodiments, the bacterial composition has ana_(w) less than 0.3 at 20° C. In embodiments, the bacterial compositionhas an a_(w) less than about 0.2 at 20° C. In embodiments, the bacterialcomposition has an a_(w) less than 0.2 at 20° C. In embodiments, thebacterial composition has an a_(w) less than about 0.1 at 20° C. Inembodiments, the bacterial composition has an a_(w) less than 0.1 at 20°C.

A “microbial composition” as provided herein refers to a compositionincluding a bacterial population that comprises, consists essentiallyof, or consists of any 1, 2, 3, 4, 5, 6, 7, or 8 of Lactobacillus sp.,Faecalibacterium sp., Akkermansia sp., Myxococcus sp., Cystobacter sp.,Pediococcus sp., Bifidobacterium sp., and Clostridium sp. Inembodiments, the bacterial population comprises Lactobacillus sp. andFaecalibacterium prausnitzii. In embodiments, the bacterial populationcomprises Lactobacillus sp. and Akkermansia muciniphila. In embodiments,the bacterial population comprises Lactobacillus sp., and Myxococcusxanthus. In embodiments, the bacterial population comprisesLactobacillus sp. and Cystobacter fuscus. In embodiments, the bacterialpopulation comprises Lactobacillus sp. and Pediococcus pentosaceus,Pediococcus acidilactici, Pediococcus damnosus, Pediococcusethanolidurans, or Pediococcus parvulus. In embodiments, the bacterialpopulation comprises Lactobacillus sp. and Bifidobacterium bifidum,Bifidobacterium pseudolongum, Bifidobacterium saeculare, orBifidobacterium subtile. In embodiments, the bacterial populationcomprises Lactobacillus sp. and Clostridium hiranonis. In embodiments,the Lactobacillus sp. is Lactobacillus johnsonii, Lactobacillusrhamnosus, Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillusacidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillusbrevis, Lactobacillus coleohominis, Lactobacillus crispatus,Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillusdiolivorans, Lactobacillus farraginis, Lactobacillus fermentum,Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillushelveticus, Lactobacillus hilgardii, Lactobacillus intestinalis,Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacilluskefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillusmanihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillusoligofermentans, Lactobacillus panis, Lactobacillus pantheris,Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillusparakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus,Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae,Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillussucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis. Inembodiments, the Lactobacillus sp. is Lactobacillus johnsonii. Inembodiments, the bacterial population comprises at least 1, 2, 3, 4, 5,6, 7, 8, 9, or 10, or from 1-5, 1-10, 1-5, or 1-20 of any combination ofthe following: Lactobacillus johnsonii. Lactobacillus rhamnosus,Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillusacidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillusbrevis, Lactobacillus coleohominis, Lactobacillus crispatus,Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillusdiolivorans, Lactobacillus farraginis, Lactobacillus fermentum,Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillushelveticus, Lactobacillus hilgardii, Lactobacillus intestinalis,Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacilluskefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillusmanihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillusoligofermentans. Lactobacillus panis, Lactobacillus pantheris,Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillusparakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus,Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae,Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillussucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus vini, Lactococcus garvieae, Lactococcus lactis,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus, Cystobacter fuscus, Pediococcus pentosaceus, Pediococcusacidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, andPediococcus parvulus. In embodiments, the bacterial population includesLactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansiamuciniphila, Myxococcus xanthus, and/or Pediococcus pentosaceus. In someembodiments, a microbial composition comprises one or more bacterialcells of the bacterial type Lactobacillus johnsonii, Faecalibacteriumprausnitzii, Akkermansia muciniphila, Myxococcus xanthus or Pediococcuspentosaceus. In embodiments, the composition includes Lactobacillusjohnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila,Myxococcus xanthus and Pediococcus pentosaceus. In embodiments, thecomposition includes Lactobacillus johnsonii, Faecalibacteriumprausnitzii, Akkermansia muciniphila, Myxococcus xanthus or Pediococcuspentosaceus. In embodiments, the bacteria are isolated. As used herein,a “type” or more than one “types” of bacteria may be differentiated atthe genus level, the species, level, the sub-species level, the strainlevel or by any other taxonomic method described herein and otherwiseknown in the art.

In embodiments, the composition includes an effective amount ofLactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansiamuciniphila, Myxococcus xanthus and Pediococcus pentosaceus. Inembodiments, the composition includes an effective amount ofLactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansiamuciniphila, Myxococcus xanthus or Pediococcus pentosaceus. Inembodiments, the composition consists essentially of an effective amountof Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansiamuciniphila, Myxococcus xanthus and Pediococcus pentosaceus. Inembodiments, the composition consists of an effective amount ofLactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansiamuciniphila, Myxococcus xanthus and Pediococcus pentosaceus. Where amicrobial composition “consists essentially of” Lactobacillus johnsonii,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus and Pediococcus pentosaceus, other agents may be included thatdo not interfere with the operation or basic and novel characteristicsof the microbial composition.

An “effective amount” is an amount sufficient to accomplish a statedpurpose (e.g. achieve the effect for which it is administered, treat adisease, reduce enzyme activity, reduce one or more symptoms of adisease or condition). An example of an “effective amount” is an amountsufficient to contribute to the treatment, prevention, or reduction of asymptom or symptoms of a disease, which could also be referred to as a“therapeutically effective amount.” Thus, an “effective amount” or“therapeutically effective amount” as provided herein refers to theamount of a bacterial population (e.g., a bacterial populationcomprising one or more species or strains of bacteria, such as abacterial population comprising Lactobacillus johnsonii,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus, and/or Pediococcus pentosaceus) required to ameliorate orprevent the symptoms of a disease (e.g., dysbiosis, an infection, or aninflammatory disease) relative to an untreated patient. In embodiments,the microbial composition does not include Lactobacillus rhamnosus.

A “reduction” of a symptom or symptoms (and grammatical equivalents ofthis phrase) means decreasing of the severity or frequency of thesymptom(s), or elimination of the symptom(s). A “prophylacticallyeffective amount” of a drug is an amount of a drug that, whenadministered to a subject, will have the intended prophylactic effect,e.g., preventing or delaying the onset (or reoccurrence) of an injury,disease, pathology or condition, or reducing the likelihood of the onset(or reoccurrence) of a disease, pathology, or condition, or theirsymptoms. The full prophylactic effect does not necessarily occur byadministration of one dose, and may occur only after administration of aseries of doses. Thus, a prophylactically effective amount may beadministered in one or more administrations. An “activity decreasingamount,” as used herein, refers to an amount of antagonist required todecrease the activity of an enzyme or protein relative to the absence ofthe antagonist. A “function disrupting amount,” as used herein, refersto the amount of antagonist required to disrupt the function of anenzyme or protein relative to the absence of the antagonist. Guidancecan be found in the literature for appropriate dosages for given classesof pharmaceutical products. For example, for the given parameter, aneffective amount will show an increase or decrease of at least 50%,100%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, or at least 100%.Efficacy can also be expressed as “-fold” increase or decrease. Forexample, a therapeutically effective amount can have at least a1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effect over a control. Theexact amounts will depend on the purpose of the treatment, and will beascertainable by one skilled in the art using known techniques (see,e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd,The Art, Science and Technology of Pharmaceutical Compounding (1999);Pickar, Dosage Calculations (1999); and Remington: The Science andPractice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott,Williams & Wilkins).

In embodiments, a composition provided herein may be administered orallyand include live microorganisms from 10³ to 10¹⁵ colony forming units(cfu)/g. In embodiments, the composition includes 10⁴ to 10¹⁵ cfu/g. Inembodiments, the composition includes 10⁵ to 10¹⁵ cfu/g. In embodiments,the composition includes 10⁶ to 10¹⁵ cfu/g. In embodiments, thecomposition includes 10⁷ to 10¹⁵ cfu/g. In embodiments, the compositionincludes 10⁸ to 10¹⁵ cfu/g. In embodiments, the composition includes 10⁹to 10¹⁵ cfu/g. In embodiments, the composition includes 10¹⁰ to 10¹⁵cfu/g. In embodiments, the composition includes 10⁸ to 10¹⁵ cfu/g. Inembodiments, the composition includes 10¹² to 10¹⁵ cfu/g. Inembodiments, the composition includes 10¹³ to 10¹⁵ cfu/g. Inembodiments, the composition includes 10¹⁴ to 10¹⁵ cfu/g. Inembodiments, the composition includes from 10³ to 10¹⁵ cfu. Inembodiments, the composition includes 10⁴ to 10¹⁵ cfu. In embodiments,the composition includes 10⁵ to 10¹⁵ cfu. In embodiments, thecomposition includes 10⁶ to 10¹⁵ cfu. In embodiments, the compositionincludes 10⁷ to 10¹⁵ cfu. In embodiments, the composition includes 0l to10¹⁵ cfu. In embodiments, the composition includes 10⁹ to 10¹⁵ cfu. Inembodiments, the composition includes 10¹⁰ to 10¹⁵ cfu. In embodiments,the composition includes 10¹¹ to 10¹⁵ cfu. In embodiments, thecomposition includes 10¹² to 10¹⁵ cfu. In embodiments, the compositionincludes 10¹³ to 10¹⁵ cfu. In embodiments, the composition includes 10¹⁴to 10¹⁵ cfu.

In embodiments, a composition provided herein may be administered orallyand include live microorganisms from 10³ to 10¹⁴ colony forming units(cfu)/g. In embodiments, the composition includes 10⁴ to 10¹⁴ cfu/g. Inembodiments, the composition includes 10⁵ to 10¹⁴ cfu/g. In embodiments,the composition includes 10⁶ to 10¹⁴ cfu/g. In embodiments, thecomposition includes 10⁷ to 10¹⁴ cfu/g. In embodiments, the compositionincludes 10⁸ to 10¹⁴ cfu/g. In embodiments, the composition includes 10⁹to 10¹⁴ cfu/g. In embodiments, the composition includes 10¹⁰ to 10¹⁴cfu/g. In embodiments, the composition includes 10¹¹ to 10¹⁴ cfu/g. Inembodiments, the composition includes 10¹² to 10¹⁴ cfu/lg. Inembodiments, the composition includes 10¹³ to 10¹⁴ cfu/g. Inembodiments, the composition includes from 10³ to 10¹⁴ cfu. Inembodiments, the composition includes 10⁴ to 10¹⁴ cfu. In embodiments,the composition includes 10⁵ to 10¹⁴ cfu. In embodiments, thecomposition includes 10⁶ to 10¹⁴ cfu. In embodiments, the compositionincludes 10⁷ to 10¹⁴ cfu. In embodiments, the composition includes 10⁸to 10¹⁴ cfu. In embodiments, the composition includes 10⁹ to 10¹⁴ cfu.In embodiments, the composition includes 10¹⁰ to 10¹⁴ cfu. Inembodiments, the composition includes 10¹¹ to 10¹⁴ cfu. In embodiments,the composition includes 10¹² to 10¹⁴ cfu. In embodiments, thecomposition includes 10¹³ to 10¹⁴ cfu.

In embodiments, a composition provided herein may be administered orallyand include live microorganisms from 10³ to 10¹³ colony forming units(cfu)/g. In embodiments, the composition includes 10⁴ to 10¹³ cfu/g. Inembodiments, the composition includes 10⁵ to 10¹³ cfu/g. In embodiments,the composition includes 10⁶ to 10¹³ cfu/g. In embodiments, thecomposition includes 10⁷ to 10¹³ cfu/g. In embodiments, the compositionincludes 10⁸ to 10¹³ cfu/g. In embodiments, the composition includes 10⁹to 10¹³ cfu/g. In embodiments, the composition includes 10¹⁰ to 10¹³cfu/g. In embodiments, the composition includes 10¹¹ to 10¹³ cfu/g. Inembodiments, the composition includes 10¹² to 10¹³ cfu/g. Inembodiments, the composition includes from 10³ to 10¹³ cfu. Inembodiments, the composition includes 10⁴ to 10¹³ cfu. In embodiments,the composition includes 10⁵ to 10¹³ cfu. In embodiments, thecomposition includes 10⁶ to 10¹³ cfu. In embodiments, the compositionincludes 10⁷ to 10¹³ cfu. In embodiments, the composition includes 10⁸to 10¹³ cfu. In embodiments, the composition includes 10⁹ to 10¹³ cfu.In embodiments, the composition includes 10¹⁰ to 10¹³ cfu. Inembodiments, the composition includes 10¹¹ to 10¹³ cfu. In embodiments,the composition includes 10¹² to 10¹³ cfu.

In embodiments, a composition provided herein may be administered orallyand include live microorganisms from 10³ to 10¹² colony forming units(cfu)/g. In embodiments, the composition includes 10⁴ to 10¹² cfu/g. Inembodiments, the composition includes 10⁵ to 10¹² cfu/g. In embodiments,the composition includes 10⁶ to 10¹² cfu/g. In embodiments, thecomposition includes 10⁷ to 10¹² cfu/g. In embodiments, the compositionincludes 10⁸ to 10¹² cfu/g. In embodiments, the composition includes 10⁹to 10¹² cfu/g. In embodiments, the composition includes 10¹⁰ to 10¹²cfu/g. In embodiments, the composition includes 10¹¹ to 10¹² cfu/g. Inembodiments, the composition includes from 10³ to 10¹² cfu. Inembodiments, the composition includes 10⁴ to 10¹² cfu/g. In embodiments,the composition includes 10⁵ to 10¹² cfu. In embodiments, thecomposition includes 10⁶ to 10¹² cfu. In embodiments, the compositionincludes 10⁷ to 10¹² cfu. In embodiments, the composition includes 10⁸to 10¹² cfu. In embodiments, the composition includes 10⁹ to 10¹² cfu.In embodiments, the composition includes 10¹⁰ to 10¹² cfu. Inembodiments, the composition includes 10¹¹ to 10¹² cfu.

In embodiments, a composition provided herein may be administered orallyand include live microorganisms from 10³ to 10¹¹ colony forming units(cfu)/g. In embodiments, the composition includes 10⁴ to 10¹¹ cfu/g. Inembodiments, the composition includes 10⁵ to 10¹¹ cfu/g. In embodiments,the composition includes 10⁶ to 10¹¹ cfu/g. In embodiments, thecomposition includes 10⁷ to 10¹¹ cfu/g. In embodiments, the compositionincludes 10⁸ to 10¹¹ cfu/g. In embodiments, the composition includes 10⁹to 10¹¹ cfu/g. In embodiments, the composition includes from 10³ to 10¹¹cfu. In embodiments, the composition includes 10⁴ to 10¹¹ cfu. Inembodiments, the composition includes 10⁵ to 10¹¹ cfu. In embodiments,the composition includes 10⁶ to 10¹¹ cfu. In embodiments, thecomposition includes 10⁷ to 10¹¹ cfu. In embodiments, the compositionincludes 10 to 10¹¹ cfu. In embodiments, the composition includes 10⁹ to10¹¹ cfu.

In embodiments, a composition provided herein may be administered orallyand include live microorganisms from 10³ to 10¹⁰ colony forming units(cfu)/g. In embodiments, the composition includes 10⁴ to 10¹⁰ cfu/g. Inembodiments, the composition includes 10⁵ to 10¹⁰ cfu/g. In embodiments,the composition includes 10⁶ to 10¹⁰ cfu/lg. In embodiments, thecomposition includes 10⁷ to 10¹⁰ cfu/g. In embodiments, the compositionincludes 10⁸ to 10¹⁰ cfu/g. In embodiments, the composition includes 10⁹to 10¹⁰ cfu/g. In embodiments, the composition includes from 10³ to 10¹⁰cfu. In embodiments, the composition includes 10⁴ to 10¹⁰ cfu. Inembodiments, the composition includes 10⁵ to 10¹⁰ cfu. In embodiments,the composition includes 10⁶ to 10¹⁰ cfu. In embodiments, thecomposition includes 10⁷ to 10¹⁰ cfu. In embodiments, the compositionincludes 10⁸ to 10¹⁰ cfu. In embodiments, the composition includes 10⁹to 10¹⁰ cfu.

In embodiments, a composition provided herein may be administered orallyand include live microorganisms from 10³ to 10⁹ colony forming units(cfu)/g. In embodiments, the composition includes 10⁴ to 10⁹ cfu/g. Inembodiments, the composition includes 10⁵ to 10⁹ cfu/g. In embodiments,the composition includes 10⁶ to 10⁹ cfu/g. In embodiments, thecomposition includes 10⁷ to 10⁹ cfu/g. In embodiments, the compositionincludes 10⁸ to 10⁹ cfu/g. In embodiments, the composition comprisesfrom 10³ to 10⁹ cfu. In embodiments, the composition includes 10⁴ to 10⁹cfu. In embodiments, the composition includes 10⁵ to 10⁹ cfu. Inembodiments, the composition includes 10⁶ to 10⁹ cfu. In embodiments,the composition includes 10⁷ to 10⁹ cfu. In embodiments, the compositionincludes 10⁸ to 10⁹ cfu.

In embodiments, a composition provided herein may be administered orallyand include live microorganisms from 10³ to 10⁸ colony forming units(cfu)/g. In embodiments, the composition includes 10⁴ to 10 cfu/g. Inembodiments, the composition includes 10⁵ to 10⁸ cfu/g. In embodiments,the composition includes 10⁶ to 10⁸ cfu/lg. In embodiments, thecomposition includes 10⁷ to 10⁸ cfu/g. In embodiments, the compositionincludes from 10³ to 10⁸ cfu. In embodiments, the composition includes10⁴ to 10⁸ cfu. In embodiments, the composition includes 10⁵ to 10⁸ cfu.In embodiments, the composition includes 10⁶ to 10⁸ cfu. In embodiments,the composition includes 10⁷ to 10⁸ cfu.

In embodiments, a composition provided herein may be administered orallyand include live microorganisms from 10³ to 10⁷ colony forming units(cfu)/g. In embodiments, the composition includes 10⁴ to 10⁷ cfu/g. Inembodiments, the composition includes 10⁵ to 10⁷ cfu/g. In embodiments,the composition includes 10⁶ to 10⁷ cfu/g. In embodiments, thecomposition includes from 10³ to 10⁷ cfu. In embodiments, thecomposition includes 10⁴ to 10⁷ cfu. In embodiments, the compositionincludes 10⁵ to 10⁷ cfu. In embodiments, the composition includes 10⁶ to10⁷ cfu.

It is understood that the amount of colony forming units (cfu)/g and cfuas provided herein may refer to the amount of each bacterial speciesstrain administered (individually) or the total cfu/g or cfu for abacterial population.

The proportion or concentration of the compositions of the invention ina pharmaceutical composition can vary depending upon a number of factorsincluding dosage, chemical characteristics (e.g., hydrophobicity), andthe route of administration. For example, the defined microbialcomposition can be provided in a capsule containing from about 0.005 mgto about 1000 mg for oral administration. Alternatively or in addition,the dosage can be expressed as cfu or cfu/g of bacteria (e.g., of dryweight when expressed as cfu/g) as described above. In embodiments, thedosage may vary, but can range from the equivalent of about 10² to about10¹⁵ cfu/g, e.g., 1×10² cfu/g, 5×10² cfu/g, 1×10³ cfu/g, 5×10³ cfu/g,1×10⁴ cfu/g, 5×10⁴ cfu/g, 1×10⁵ cfu/g, 5×10⁵ cfu/g, 1×10⁶ cfu/g, 5×10⁶cfu/g, 1×10⁷ cfu/g, 5×10⁷ cfu/g, 1×10⁸ cfu/g, 5×10⁸ cfu/g, 1×10⁹ cfu/g,5×10⁹ cfu/g, 1×10¹⁰ cfu/g, 5×10¹⁰ cfu/g, 1×10¹¹ cfu/g, 5×10¹¹ cfu/g, or1×10¹² cfu/g of dry weight. In embodiments, Lactobacillus johnsonii.Faecalibacterium prausnitzii, Akkermansia muciniphila. Myxococcusxanthus or Pediococcus pentosaceus are administered at any one of 10³,10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10 ¹², 10¹³, 10¹⁴, or 10¹⁵colony forming units (cfu)/g of dry weight, or total cfu, individuallyor total. In embodiments, the composition includes Lactobacillusjohnsonii at about 10⁷ colony forming units (cfu)/g or a total of 10⁷cfu. In embodiments, the composition includes Akkermansia muciniphila atabout 10⁷ colony forming units (cfu)/g or a total of 10⁷ cfu. Inembodiments, the composition includes Myxococcus xanthus at about 10⁷colony forming units (cfu)/g or a total of 10⁷ cfu. In embodiments, thecomposition includes Pediococcus pentosaceus at about 10⁷ colony formingunits (cfu)/g or a total of 10⁷ cfu. In embodiments, the compositionincludes Faecalibacterium prausnitzii at about 10 colony forming units(cfu)/g or a total of 10 cfu. In embodiments, the composition includeslive microorganisms (e.g., Lactobacillus johnsonii, Faecalibacteriumprausnitzii, Akkermansia muciniphila, Myxococcus xanthus or Pediococcuspentosaceus) per gram of composition, or equivalent doses calculated forinactivated or dead microorganisms or for microorganism fractions or forproduced metabolites.

In embodiments, Lactobacillus johnsonii as provided herein refers to oneor more isolated bacterial cells of a strain cultured from murineintestines using Lactobacillus isolation media (deMan, Rogose and Sharpeagar). Non-limiting examples of Lactobacillus johnsonii include strainsdeposited with ATCC under Accession Nos. 11506 and 53672.

In embodiments, Lactobacillus rhamnosus as provided herein refers to oneor more isolated bacterial cells of a bacterial strain having all theidentifying characteristics of a strain deposited with ATCC as AccessionNo. 53103; variants of the strain deposited with ATCC as Accession No.53103 having all the identifying characteristics of the ATCC No. 53103strain; and mutants of the strain deposited with ATCC as Accession No.53103 having all the identifying characteristics of the ATCC No. 53103strain.

In embodiments, Faecalibacterium prausnitzii as provided herein refersto one or more isolated bacterial cells of a bacterial strain having allthe identifying characteristics of a strain deposited with ATCC asAccession No. 27766; variants of the strain deposited with ATCC asAccession No. 27766 having all the identifying characteristics of theATCC No. 27766 strain; and mutants of the strain deposited with ATCC asAccession No. 27766 having all the identifying characteristics of theATCC No. 27766 strain.

In embodiments, Akkermansia muciniphila as provided herein refers to oneor more isolated bacterial cells of a bacterial strain having all theidentifying characteristics of a strain deposited with ATCC as AccessionNo. BAA-835; variants of the strain deposited with ATCC as Accession No.BAA-835 having all the identifying characteristics of the ATCC No.BAA-835 strain; and mutants of the strain deposited with ATCC asAccession No. BAA-835 having all the identifying characteristics of theATCC No. BAA-835 strain.

In embodiments, Myxococcus xanthus as provided herein refers to one ormore isolated bacterial cells of a bacterial strain having all theidentifying characteristics of a strain deposited with ATCC as AccessionNo. 25232; variants of the strain deposited with ATCC as Accession No.25232 having all the identifying characteristics of the ATCC No. 25232strain; and mutants of the strain deposited with ATCC as Accession No.25232 having all the identifying characteristics of the ATCC No. 25232strain.

In embodiments, Pediococcus pentosaceus as provided herein refers to oneor more isolated bacterial cells of a bacterial strain having all theidentifying characteristics of a strain deposited with ATCC as AccessionNo. 25744; variants of the strain deposited with ATCC as Accession No.25744 having all the identifying characteristics of the ATCC No. 25744strain; and mutants of the strain deposited with ATCC as Accession No.25744 having all the identifying characteristics of the ATCC No. 25744strain.

In embodiments, the composition is effective to increase ananti-inflammatory metabolite. In embodiments, the Lactobacillusjohnsonii is effective to increase an anti-inflammatory metabolite. Inembodiments, the Faecalibacterium prausnitzii is effective to increasean anti-inflammatory metabolite. In embodiments, the Akkermansiamuciniphila is effective to increase an anti-inflammatory metabolite. Inembodiments, the Myxococcus xanthus is effective to increase ananti-inflammatory metabolite. In embodiments, the Pediococcuspentosaceus is effective to increase an anti-inflammatory metabolite. A“metabolite” as provided herein refers to intermediates and products ofthe metabolism of a bacterial cell, wherein the bacterial cell resideswithin the gut of a mammal. The term metabolite also includesintermediates and products formed by a mammalian cell. Non-limitingexamples of metabolites include amino acids, alcohols, vitamins,polyols, organic acids, nucleotides (e.g. inosine-5′-monophosphate andguanosine-5′-monophosphate), lipids, carbohydrates, peptides andproteins. An “anti-inflammatory metabolite” as provided herein refers toa metabolite produced by a cell (e.g., bacterial cell, mammalian cell)and capable of inhibiting inflammation. As defined herein, the term“inhibition”, “inhibit”, “inhibiting” and the like in reference to aprotein-anti-inflammatory metabolite interaction means negativelyaffecting (e.g., decreasing) the activity or function of the protein(e.g., decreasing the activity of an inflammatory metabolite) relativeto the activity or function of the protein in the absence of theinhibitor (e.g., anti-inflammatory metabolite). The term “inhibiting”includes, at least in part, partially or totally blocking stimulation,decreasing, preventing, or delaying activation, or inactivating,desensitizing, or down-regulating signal transduction, gene expression,enzymatic activity or protein expression (e.g., inflammatory metabolite)necessary for inflammation. In some embodiments inhibition refers toreduction of a disease or symptoms of disease (e.g., inflammation).Similarly an “inhibitor” is a compound (e.g., metabolite) that inhibitsinflammation, e.g., by binding, partially or totally blocking,decreasing, preventing, delaying, inactivating, desensitizing, ordown-regulating inflammatory metabolite activity. A metabolite capableof inhibiting or decreasing inflammation as provided herein refers to asubstance that results in a detectably lower activity level ofinflammation of as compared to a control. The decreased activity can be10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less than that in acontrol. In certain instances, the decrease is 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold, 10-fold, or less in comparison to a control.

In embodiments, the anti-inflammatory metabolite is a microbial lipid ora microbial carbohydrate. In embodiments, the anti-inflammatorymetabolite is a microbial lipid. In embodiments, the anti-inflammatorymetabolite is a phospholipid. In embodiments, the anti-inflammatorymetabolite is a poly-unsaturated fatty acid. In embodiments, theanti-inflammatory metabolite is microbial carbohydrate. In embodiments,the anti-inflammatory metabolite is itoconate. In embodiments, theanti-inflammatory metabolite is n-acetylglucosamine. In embodiments, theanti-inflammatory metabolite is n-acetylgalactosamine. In embodiments,the anti-inflammatory metabolite is fucosyllactose. In embodiments, theanti-inflammatory metabolite is an amino acid. In embodiments, theanti-inflammatory metabolite is tryptophan.

In embodiments, the composition is effective to decrease apro-inflammatory metabolite. In embodiments, the composition iseffective to decrease pro-inflammatory metabolite. In embodiments, theLactobacillus johnsonii is effective to decrease a pro-inflammatorymetabolite. In embodiments, the Faecalibacterium prausnitzii iseffective to decrease a pro-inflammatory metabolite. In embodiments, theAkkermansia muciniphila is effective to decrease a pro-inflammatorymetabolite. In embodiments, the Myxococcus xanthus is effective todecrease pro-inflammatory metabolite. In embodiments, the Pediococcuspentosaceus is effective to decrease pro-inflammatory metabolite. A“pro-inflammatory metabolite” as provided herein refers to a metaboliteproduced by a cell (e.g., bacterial cell, mammalian cell) and capable ofincreasing inflammation. A metabolite capable of increasing inflammationas provided herein refers to a substance that results in a detectablyhigher level of inflammation as compared to a control. The increasedactivity can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 900/or more thanthat in a control. In certain instances, the increase is 1.5-fold,2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more in comparison to acontrol.

In embodiments, the pro-inflammatory metabolite is a microbial lipid, amicrobial carbohydrate or a microbial amino acid. In embodiments, thepro-inflammatory metabolite is a microbial lipid. In embodiments, thepro-inflammatory metabolite is dihydroxyoctadec-12-enoic acid, cholateor methylmalonate. In embodiments, the pro-inflammatory metabolite is amicrobial carbohydrate. In embodiments, the pro-inflammatory metaboliteis n-acetylymuramate, lactobionate or maltotriose. In embodiments, thepro-inflammatory metabolite is a microbial amino acid. In embodiments,the pro-inflammatory metabolite is ornithine or taurine.

The compositions provided herein may include metabolically activebacteria or metabolically inactive bacteria or fractions thereof. Inembodiments, the Lactobacillus johnsonii, Faecalibacterium prausnitzii,the Akkermansia muciniphila, the Myxococcus xanthus and the Pediococcuspentosaceus are metabolically active. In embodiments, the saidLactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansiamuciniphila, Myxococcus xanthus and Pediococcus pentosaceus aremetabolically inactive. Metabolically active bacteria are capable ofdividing and produce metabolites such as carbohydrates, lipids or aminoacids. In contrast metabolically inactive bacteria do not divide orproduce metabolites.

III. Pharmaceutical Compositions

As described herein, the microbial compositions provided herein mayinclude a bacterial population that comprises, consists essentially of,or consists of any 1, 2, 3, 4, 5, 6, 7, or 8 of Lactobacillus sp.,Faecalibacterium sp., Akkermansia sp., Myxococcus sp., Cystobacter sp.,Pediococcus sp., Bifidobacterium sp., and Clostridium sp. Inembodiments, the bacterial population comprises Lactobacillus sp. andFaecalibacterium prausnitzii. In embodiments, the bacterial populationcomprises Lactobacillus sp. and Akkermansia muciniphila. In embodiments,the bacterial population comprises Lactobacillus sp., and Myxococcusxanthus. In embodiments, the bacterial population comprisesLactobacillus sp. and Cystobacter fuscus. In embodiments, the bacterialpopulation comprises Lactobacillus sp. and Pediococcus pentosaceus,Pediococcus acidilactici, Pediococcus damnosus, Pediococcusethanolidurans, or Pediococcus parvulus. In embodiments, the bacterialpopulation comprises Lactobacillus sp. and Bifidobacterium bifidum,Bifidobacterium pseudolongum, Bifidobacterium saeculare, orBifidobacterium subtile. In embodiments, the bacterial populationcomprises Lactobacillus sp. and Clostridium hiranonis. In embodiments,the Lactobacillus sp. is Lactobacillus johnsonii, Lactobacillusrhamnosus, Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillusacidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillusbrevis, Lactobacillus coleohominis, Lactobacillus crispatus,Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillusdiolivorans, Lactobacillus farraginis, Lactobacillus fermentum,Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillushelveticus, Lactobacillus hilgardii, Lactobacillus intestinalis,Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacilluskefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillusmanihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillusoligofermentans, Lactobacillus panis, Lactobacillus pantheris,Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillusparakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus,Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae,Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillussucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis. Inembodiments, the Lactobacillus sp. is Lactobacillus johnsonii. Inembodiments, the bacterial population comprises at least 1, 2, 3, 4, 5,6, 7, 8, 9, or 10, or from 1-5, 1-10, 1-5, or 1-20 of any combination ofthe following: Lactobacillus johnsonii, Lactobacillus rhamnosus,Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillusacidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillusbrevis, Lactobacillus coleohominis, Lactobacillus crispatus,Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillusdiolivorans, Lactobacillus farraginis, Lactobacillus fermentum,Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillushelveticus, Lactobacillus hilgardii, Lactobacillus intestinalis,Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacilluskefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillusmanihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillusoligofermentans, Lactobacillus panis, Lactobacillus pantheris,Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillusparakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus,Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae,Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillussucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus vini, Lactococcus garvieae, Lactococcus lactis,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus, Cystobacter fuscus, Pediococcus pentosaceus, Pediococcusacidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, andPediococcus parvulus. In embodiments, the bacterial population includesLactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansiamuciniphila, Myxococcus xanthus, and/or Pediococcus pentosaceus. In someembodiments, a microbial composition comprises one or more bacterialcells of the bacterial type Lactobacillus johnsonii. Faecalibacteriumprausnitzii, Akkermansia muciniphila, Myxococcus xanthus or Pediococcuspentosaceus. In embodiments, the composition includes Lactobacillusjohnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila,Myxococcus xanthus and Pediococcus pentosaceus. In embodiments, thecomposition includes Lactobacillus johnsonii, Faecalibacteriumprausnitzii, Akkermansia muciniphila, Myxococcus xanthus or Pediococcuspentosaceus. In embodiments, the bacteria are isolated bacteria.

In embodiments, the microbial composition includes a therapeuticallyeffective amount of Lactobacillus johnsonii, Faecalibacteriumprausnitzii. Akkermansia muciniphila, Myxococcus xanthus and/orPediococcus pentosaceus.

In embodiments, the microbial composition further includes apharmaceutically acceptable excipient. Thus, in one aspect apharmaceutical composition including a therapeutically effective amountof Lactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansiamuciniphila, Myxococcus xanthus, and Pediococcus pentosaceus and apharmaceutically acceptable excipient are provided.

“Pharmaceutically acceptable excipient” and “pharmaceutically acceptablecarrier” refer to a substance that aids the administration of an activeagent to and absorption by a subject and can be included in thecompositions of the present invention without causing a significantadverse toxicological effect on the patient. Non-limiting examples ofpharmaceutically acceptable excipients include water, NaCl, normalsaline solutions, lactated Ringer's, normal sucrose, normal glucose,binders, fillers, disintegrants, lubricants, coatings, sweeteners,flavors, salt solutions (such as Ringer's solution), alcohols, oils,gelatins, carbohydrates such as lactose, amylose or starch, fatty acidesters, hydroxymethycellulose, polyvinyl pyrrolidine, and colors, andthe like. Such preparations can be sterilized and, if desired, mixedwith auxiliary agents such as lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, coloring, and/or aromatic substances and the like that do notdeleteriously react with the compounds of the invention. One of skill inthe art will recognize that other pharmaceutical excipients are usefulin the present invention.

The microbial compositions provided herein including embodiments thereofmay be adminstered orally, gastrointestinally, or rectally.Administration can be in the form of a single bolus dose, or may be, forexample, by a continuous perfusion pump. In embodiments, the microbialconsortium provided herein is combined with one or more excipients, forexample, a disintegrant, a filler, a glidant, or a preservative. Inembodiments, the microbial consortium provided herein forms part of acapsule. Suitable capsules include both hard shell capsules orsoft-shelled capsules. Any lipid-based or polymer-based colloid may beused to form the capsule. Exemplary polymers useful for colloidpreparations include gelatin, plant polysaccharides or their derivativessuch as carrageenans and modified forms of starch and cellulose, e.g.,hypromellose. Optionally, other ingredients may be added to the gellingagent solution, for example plasticizers such as glycerin and/orsorbitol to decrease the capsule's hardness, coloring agents,preservatives, disintegrants, lubricants and surface treatment.

The microbial compositions can be formulated in a unit dosage form, eachdosage containing, for example, from about 0.005 mg to about 2000 mg ofa defined microbial consortium having minimal urease activity per dose.The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of active material calculatedto produce the desired therapeutic effect, in association with asuitable pharmaceutical excipient. For preparing solid compositions suchas tablets, the principal active ingredient is mixed with apharmaceutical excipient to form a solid preformulation compositioncontaining a homogeneous mixture of a compound of the present invention.When referring to these preformulation compositions as homogeneous, theactive ingredient is typically dispersed evenly throughout thecomposition so that the composition can be readily subdivided intoequally effective unit dosage forms such as tablets, pills and capsules.This solid preformulation is then subdivided into unit dosage forms ofthe type described above containing from, for example, 0.005 mg to about1000 mg of the microbial composition provided herein.

The microbial compositions can be formulated in a unit dosage form, eachdosage containing, for example, from about 0.1 mg to about 50 mg, fromabout 0.1 mg to about 40 mg, from about 0.1 mg to about 20 mg from about0.1 mg to about 10 mg, from about 0.2 mg to about 20 mg, from about 0.3mg to about 15 mg, from about 0.4 mg to about 10 mg, from about 0.5 mgto about 1 mg; from about 0.5 mg to about 100 mg, from about 0.5 mg toabout 50 mg, from about 0.5 mg to about 30 mg, from about 0.5 mg toabout 20 mg, from about 0.5 mg to about 10 mg, from about 0.5 mg toabout 5 mg; from about 1 mg from to about 50 mg, from about 1 mg toabout 30 mg, from about 1 mg to about 20 mg, from about 1 mg to about 10mg, from about 1 mg to about 5 mg; from about 5 mg to about 50 mg, fromabout 5 mg to about 20 mg, from about 5 mg to about 10 mg; from about 10mg to about 100 mg, from about 20 mg to about 200 mg, from about 30 mgto about 150 mg, from about 40 mg to about 100 mg, from about 50 mg toabout 100 mg of Lactobacillus sp. (e.g., Lactobacillus johnsonii),Faecalibacterium sp. (Faecalibacterium prausnitzii), Akkermansia sp.(e.g., Akkermansia muciniphila), Myxococcus sp. (e.g., Myxococcusxanthus) and/or Pediococcus sp. (e.g., Pediococcus pentosaceus)individually or combined.

In some embodiments, tablets or pills of the present invention can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer which serves to resist disintegration inthe stomach and permit the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol, and cellulose acetate.

The liquid forms in which the compositions of the present invention canbe incorporated for administration orally or by injection includeaqueous solutions, suitably flavored syrups, aqueous or oil suspensions,and flavored emulsions with edible oils such as cottonseed oil, sesameoil, coconut oil, or peanut oil, as well as elixirs and similarpharmaceutical vehicles.

IV. Methods of Treatment

According to the methods provided herein, the subject is administered aneffective amount of one or more of the agents provided herein. The termseffective amount and effective dosage are used interchangeably. The termeffective amount is defined as any amount necessary to produce a desiredphysiologic response (e.g., reduction of inflammation, infection, ordysbiosis). Effective amounts and schedules for administering the agentmay be determined empirically by one skilled in the art. The dosageranges for administration are those large enough to produce the desiredeffect in which one or more symptoms of the disease or disorder areaffected (e.g., reduced or delayed). The dosage should not be so largeas to cause substantial adverse side effects, such as unwantedcross-reactions, anaphylactic reactions, and the like. Generally, thedosage will vary with the age, condition, sex, type of disease, theextent of the disease or disorder, route of administration, or whetherother drugs are included in the regimen, and can be determined by one ofskill in the art. The dosage can be adjusted by the individual physicianin the event of any contraindications. Dosages can vary and can beadministered in one or more dose administrations daily, for one orseveral days. Guidance can be found in the literature for appropriatedosages for given classes of pharmaceutical products. For example, forthe given parameter, an effective amount will show an increase ordecrease of at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%,90%, or at least 100%. Efficacy can also be expressed as “-fold”increase or decrease. For example, a therapeutically effective amountcan have at least a 1.2-fold, 1.5-fold, 2-fold, 5-fold, or more effectover a control. The exact dose and formulation will depend on thepurpose of the treatment, and will be ascertainable by one skilled inthe art using known techniques (see, e.g., Lieberman, PharmaceuticalDosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technologyof Pharmaceutical Compounding (1999); Remington: The Science andPractice of Pharmacy, 20th Edition, Gennaro, Editor (2003), and Pickar,Dosage Calculations (1999)).

For prophylactic use, a therapeutically effective amount of themicrobial composition described herein are administered to a subjectprior to or during early onset (e.g., upon initial signs and symptoms ofan autoimmune disease). Therapeutic treatment involves administering toa subject a therapeutically effective amount of the agents describedherein after diagnosis or development of disease. Thus, in anotheraspect, a method of treating a disease (e.g., an inflammatory disease,an infection, or dysbiosis) in a subject in need thereof is provided.

The terms “subject,” “patient,” “individual,” etc. are not intended tobe limiting and can be generally interchanged. That is, an individualdescribed as a “patient” does not necessarily have a given disease, butmay be merely seeking medical advice.

As used herein, “treating” or “treatment of” a condition, disease ordisorder or symptoms associated with a condition, disease or disorderrefers to an approach for obtaining beneficial or desired results,including clinical results. Beneficial or desired clinical results caninclude, but are not limited to, alleviation or amelioration of one ormore symptoms or conditions, diminishment of extent of condition,disorder or disease, stabilization of the state of condition, disorderor disease, prevention of development of condition, disorder or disease,prevention of spread of condition, disorder or disease, delay or slowingof condition, disorder or disease progression, delay or slowing ofcondition, disorder or disease onset, amelioration or palliation of thecondition, disorder or disease state, and remission, whether partial ortotal. “Treating” can also mean prolonging survival of a subject beyondthat expected in the absence of treatment. “Treating” can also meaninhibiting the progression of the condition, disorder or disease,slowing the progression of the condition, disorder or diseasetemporarily, although in some instances, it involves halting theprogression of the condition, disorder or disease permanently. As usedherein the terms treatment, treat, or treating refers to a method ofreducing the effects of one or more symptoms of a disease or conditioncharacterized by expression of the protease or symptom of the disease orcondition characterized by expression of the protease. Thus in thedisclosed method, treatment can refer to a 10%, 200, 30%, 40%, 50%, 60%,70%, 80%, 90%, or 100% reduction in the severity of an establisheddisease, condition, or symptom of the disease or condition (e.g.,inflammation, infection, or dysbiosis). For example, a method fortreating a disease is considered to be a treatment if there is a 10%reduction in one or more symptoms of the disease in a subject ascompared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%,50°, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10%and 100% as compared to native or control levels. It is understood thattreatment does not necessarily refer to a cure or complete ablation ofthe disease, condition, or symptoms of the disease or condition.Further, as used herein, references to decreasing, reducing, orinhibiting include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,90% or greater as compared to a control level and such terms can includebut do not necessarily include complete elimination.

Compositions comprising a defined microbial compositions can beadministered to the gastrointestinal tract of a subject by nasoduodenalcatheter, by enema, or by endoscopy, enteroscopy, or colonoscopy ororally in a consumable capsule or pill. In certain embodiments, thedefined microbial compositions are diluted in a suitable excipient(e.g., saline solution). In a preferred embodiment, the bacteria aredelivered in lyophilized form.

Regardless of how the compositions are formulated, the dosage requiredwill depend on the route of administration, the nature of theformulation, the nature of the subject's condition, e.g., immaturity ofthe immune system or a gastrointestinal disorder, the subject's size,weight, surface area, age, and sex, other drugs being administered, andthe judgment of the attending clinicians. In embodiments, suitabledosages are in the range of 0.01-1,000 mg/kg. Some typical dose rangesare from about 1 μg/kg to about 1 g/kg of body weight per day. Inembodiments, the dose range is from about 0.01 mg/kg to about 100 mg/kgof body weight per day. In embodiments, the dose can be, for example, 1mg/kg, 2 mg/kg, 5 mg kg, 10 mg/kg, 20 mg/kg, 50 mg/kg or 100 mg/kg.Alternatively or in addition, the dosage can be expressed as cfu or ascfu/g of dry weight. In embodiments, the dosage may vary, but can rangefrom the equivalent of about 10² to about 10¹² cfu/g, e.g., 1×10² cfu/g,5×10² cfu/g, 1×10³ cfu/g, 5×10³ cfu/g, 1×10⁴ cfu/g, 5×10⁴ cfu/g, 1×10⁵cfu/g, 5×10⁵ cfu/g, 1×10⁶ cfu/g, 5×10⁶ cfu/g, 1×10⁷ cfu/g, 5×10⁷ cfu/g,1×10⁸ cfu/g, 5×10⁸ cfu/g, 1×10⁹ cfu/g, 5×10⁹ cfu/g, 1×10¹⁰ cfu/g, 5×10¹⁰cfu/g, 1×10¹¹ cfu/g, 5×10¹¹ cfu/g, or 1×10¹² cfu/g of dry weight of anyone of the administered bacteria (individually) or of the totalpopulation of bacteria. In embodiments, the dosage can range from about10² to about 10¹² cfu, e.g., 1×10² cfu, 5×10² cfu, 1×10³ cfu, 5×10³ cfu,1×10⁴ cfu, 5×10⁴ cfu, 1×10⁵ cfu, 5×10⁵ cfu, 1×10⁶ cfu, 5×10⁶ cfu, 1×10⁷cfu, 5×10⁷ cfu, 1×10⁸ cfu, 5×10⁸ cfu, 1×10⁹ cfu, 5×10⁹ cfu, 1×10¹⁰ cfu,5×10¹⁰ cfu, 1×10¹¹ cfu, 5×10¹¹ cfu, or 1×10¹² cfu of any one of theadministered bacteria (individually) or of the total population ofbacteria.

Administrations can be single or multiple (e.g., 2- or 3-, 4-, 6-, 8-,10-, 20-, 50-, 100-, 150-, or more fold). The duration of treatment withany composition provided herein can be any length of time from as shortas one day to as long as the life span of the host (e.g., many years).For example, a composition can be administered 1, 2, 3, 4, 5, 6, or 7times a week (for, for example, 4 weeks to many months or years); once amonth (for example, three to twelve months or for many years); or once ayear for a period of 5 years, ten years, or longer. It is also notedthat the frequency of treatment can be variable. For example, thepresent compositions can be administered once (or twice, three times,etc.) daily, weekly, monthly, or yearly.

The compositions may also be administered in conjunction with othertherapeutic agents. Other therapeutic agents will vary according to theparticular disorder, but can include, for example, dietary modification,hemodialysis, therapeutic agents such as sodium benzoate, phenylacetate,arginine, or surgical remedies. Concurrent administration of two or moretherapeutic agents does not require that the agents be administered atthe same time or by the same route, as long as there is an overlap inthe time period during which the agents are exerting their therapeuticeffect. Simultaneous or sequential administration is contemplated, as isadministration on different days or weeks.

Provided herein are methods of treating and preventing inflammatorydiseases, infections (such as respiratory or gastrointestinalinfections) and dysbiosis comprising administering the bacterialpopulations or microbial compositions described herein includingembodiments thereof.

In an aspect, a method of treating or preventing dysbiosis, aninflammatory disease, or a viral respiratory infection, in a subject inneed thereof is provided.

In an aspect, a method of increasing the level of an anti-inflammatorycompound and/or decreasing the level of a pro-inflammatory compound in asubject in need thereof is provided.

In an aspect, a method of altering the metabolism of a subject in needthereof is provided.

In embodiments, the method includes administering to the subject aneffective amount of a bacterial population that comprises, consistsessentially of, or consists of, 1, 2, 3, 4, 5, 6, 7, or 8 (or at least1, 2, 3, 4, 5, 6, 7, or 8) bacterial species. In embodiments, thebacterial population comprises, consists essentially of, or consists ofany 1, 2, 3, 4, 5, 6, 7, or 8 of Lactobacillus sp., Faecalibacteriumsp., Akkermansia sp., Myxococcus sp., Cystobacter sp., Pediococcus sp.,Bifidobacterium sp., and Clostridium sp. In embodiments, the bacterialpopulation comprises Lactobacillus sp. and Faecalibacterium prausnitzii.In embodiments, the bacterial population comprises Lactobacillus sp. andAkkermansia muciniphila. In embodiments, the bacterial populationcomprises Lactobacillus sp., and Myxococcus xanthus. In embodiments, thebacterial population comprises Lactobacillus sp. and Cystobacter fuscus.In embodiments, the bacterial population comprises Lactobacillus sp. andPediococcus pentosaceus, Pediococcus acidilactici, Pediococcus damnosus,Pediococcus ethanolidurans, or Pediococcus parvulus. In embodiments, thebacterial population comprises Lactobacillus sp. and Bifidobacteriumbifidum, Bifidobacterium pseudolongum, Bifidobacterium saeculare, orBifidobacterium subtile. In embodiments, the bacterial populationcomprises Lactobacillus sp. and Clostridium hiranonis. In embodiments,the Lactobacillus sp. is Lactobacillus johnsonii, Lactobacillusrhamnosus, Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillusacidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillusbrevis, Lactobacillus coleohominis, Lactobacillus crispatus,Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillusdiolivorans, Lactobacillus farraginis, Lactobacillus fermentum,Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillushelveticus, Lactobacillus hilgardii, Lactobacillus intestinalis,Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacilluskefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillusmanihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillusoligofermentans, Lactobacillus panis, Lactobacillus pantheris,Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillusparakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus,Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae,Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillussucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis. Inembodiments, the Lactobacillus sp. is Lactobacillus johnsonii. Inembodiments, the bacterial population comprises at least 1, 2, 3, 4, 5,6, 7, 8, 9, or 10, or from 1-5, 1-10, 1-5, or 1-20 of any combination ofthe following: Lactobacillus johnsonii, Lactobacillus rhamnosus,Lactobacillus zeae, Lactobacillus acidipiscis, Lactobacillusacidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillusbrevis, Lactobacillus coleohominis, Lactobacillus crispatus,Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillusdiolivorans, Lactobacillus farraginis, Lactobacillus fermentum,Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillushelveticus, Lactobacillus hilgardii, Lactobacillus intestinalis,Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacilluskefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillusmanihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillusoligofermentans, Lactobacillus panis, Lactobacillus pantheris,Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillusparakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus,Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae,Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillussucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus vini, Lactococcus garvieae, Lactococcus lactis,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus, Cystobacter fuscus, Pediococcus pentosaceus, Pediococcusacidilactici, Pediococcus damnosus, Pediococcus ethanolidurans, andPediococcus parvulus. In embodiments, the bacterial population includesLactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansiamuciniphila, Myxococcus xanthus, and/or Pediococcus pentosaceus. Inembodiments, the bacteria are isolated bacteria.

In embodiments, the method includes administering to the subject aneffective amount of a bacterial population including Lactobacillus sp.,Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and Pediococcussp. In embodiments, (i) the Lactobacillus sp. is Lactobacillusjohnsonii; (ii) the Faecalibacterium sp., is Faecalibacteriumprausnitzii; (iii) the Akkermansia sp. is Akkermansia muciniphila; (iv)the Myxococcus sp. is Myxococcus xanthus; and (v) the Pediococcus sp. isPediococcus pentosaceus. In embodiments, (i) the Lactobacillus sp. isLactobacillus zeae, Lactobacillus acidipiscis, Lactobacillusacidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillusbrevis, Lactobacillus coleohominis, Lactobacillus crispatus,Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillusdiolivorans, Lactobacillus farraginis, Lactobacillus fermentum,Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillushelveticus, Lactobacillus hilgardii, Lactobacillus intestinalis,Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacilluskefiri, Lactobacillus lindneri, Lactobacillus mali, Lactobacillusmanihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillusoligofermentans, Lactobacillus panis, Lactobacillus pantheris,Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillusparakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus,Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae,Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillussucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis; (ii)the Faecalibacterium sp., is Faecalibacterium prausnitzii; (iii) theAkkermansia sp. is Akkermansia muciniphila; (iv) the Myxococcus sp. isMyxococcus xanthus; and (v) the Pediococcus sp. is Pediococcuspentosaceus, Pediococcus acidilactici, Pediococcus damnosus, Pediococcusethanolidurans, or Pediococcus parvulus.

In embodiments, the Myxococcus sp. is in the form of spores, vegetativebacteria, or a mixture of spores and vegetative bacteria. Inembodiments, the Myxococcus sp. is in the form of a powder comprisingspores. In embodiments, the Clostridium sp. is in the form of spores,vegetative bacteria, or a mixture of spores and vegetative bacteria. Inembodiments, the Clostridium sp. is in the form of a powder comprisingspores.

In embodiments, less than about 20, 15, 10, 9, 8, 7, or 6 differentspecies of bacteria are administered to the subject. In embodiments,less than about 20 different species of bacteria are administered to thesubject. In embodiments, less than 20 different species of bacteria areadministered to the subject. In embodiments, less than about 15different species of bacteria are administered to the subject. Inembodiments, less than 15 different species of bacteria are administeredto the subject. In embodiments, less than about 10 different species ofbacteria are administered to the subject. In embodiments, less than 10different species of bacteria are administered to the subject. Inembodiments, less than about 9 different species of bacteria areadministered to the subject. In embodiments, less than 9 differentspecies of bacteria are administered to the subject. In embodiments,less than about 8 different species of bacteria are administered to thesubject. In embodiments, less than 8 different species of bacteria areadministered to the subject. In embodiments, less than about 7 differentspecies of bacteria are administered to the subject. In embodiments,less than 7 different species of bacteria are administered to thesubject. In embodiments, less than about 6 different species of bacteriaare administered to the subject. In embodiments, less than 6 differentspecies of bacteria are administered to the subject.

In embodiments, the bacterial population forms part of a bacterialcomposition. In embodiments, the bacterial composition includes lessthan about 20, 15, 10, 9, 8, 7, or 6 species of bacteria. Inembodiments, the bacterial composition includes less than about 20species of bacteria. In embodiments, the bacterial composition includesless than 20 species of bacteria. In embodiments, the bacterialcomposition includes less than about 15 species of bacteria. Inembodiments, the bacterial composition includes less than 15 species ofbacteria. In embodiments, the bacterial composition includes less thanabout 10 species of bacteria. In embodiments, the bacterial compositionincludes less than 10 species of bacteria. In embodiments, the bacterialcomposition includes less than about 9 species of bacteria. Inembodiments, the bacterial composition includes less than 9 species ofbacteria. In embodiments, the bacterial composition includes less thanabout 8 species of bacteria. In embodiments, the bacterial compositionincludes less than 8 species of bacteria. In embodiments, the bacterialcomposition includes less than about 7 species of bacteria. Inembodiments, the bacterial composition includes less than 7 species ofbacteria. In embodiments, the bacterial composition includes less thanabout 6 species of bacteria. In embodiments, the bacterial compositionincludes less than 6 species of bacteria.

In embodiments, the bacterial composition further includes apharmaceutically acceptable excipient. In embodiments, the bacterialcomposition is not a fecal transplant. In embodiments, the bacterialcomposition is a capsule, a tablet, a suspension, a suppository, apowder, a cream, an oil, an oil-in-water emulsion, a water-in-oilemulsion, or an aqueous solution. In embodiments, the bacterialcomposition is in the form of a powder, a solid, a semi-solid, or aliquid.

In embodiments, the bacterial composition has a water activity (a_(w))less than about 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 at 20° C.In embodiments, the bacterial composition has an a_(w) less than about0.9 at 20° C. In embodiments, the bacterial composition has an a_(w)less than 0.9 at 20° C. In embodiments, the bacterial composition has ana_(w) less than about 0.8 at 20° C. In embodiments, the bacterialcomposition has an a_(w) less than 0.8 at 20° C. In embodiments, thebacterial composition has an a_(w) less than about 0.7 at 20° C. Inembodiments, the bacterial composition has an a_(w) less than 0.7 at 20°C. In embodiments, the bacterial composition has an a_(w) less thanabout 0.6 at 20° C. In embodiments, the bacterial composition has ana_(w) less than 0.6 at 20° C. In embodiments, the bacterial compositionhas an a_(w) less than about 0.5 at 20° C. In embodiments, the bacterialcomposition has an a_(w) less than 0.5 at 20° C. In embodiments, thebacterial composition has an a_(w) less than about 0.4 at 20° C. Inembodiments, the bacterial composition has an a_(w) less than 0.4 at 20°C. In embodiments, the bacterial composition has an a_(w) less thanabout 0.3 at 20° C. In embodiments, the bacterial composition has ana_(w) less than 0.3 at 20° C. In embodiments, the bacterial compositionhas an a_(w) less than about 0.2 at 20° C. In embodiments, the bacterialcomposition has an a_(w) less than 0.2 at 20° C. In embodiments, thebacterial composition has an a_(w) less than about 0.1 at 20° C. Inembodiments, the bacterial composition has an a_(w) less than 0.1 at 20°C.

In embodiments, the bacterial composition is a food or a beverage.

In embodiments, the bacterial composition is administered orally orrectally.

In embodiments, the Lactobacillus sp., the Faecalibacterium sp., theAkkermansia sp., the Myxococcus sp., and/or the Pediococcus sp. is inthe form of a powder. In embodiments, the Lactobacillus sp., theFaecalibacterium sp., the Akkermansia sp., the Myxococcus sp., and/orthe Pediococcus sp. has been lyophilized.

In embodiments, the subject is a human. In embodiments, the subjectsuffers from or resides with someone who suffers from a bacterial,viral, or fungal gastrointestinal infection.

In embodiments, the subject has an inflammatory disease. In embodiments,the subject is at risk of suffering from an inflammatory disease. Inembodiments, the subject has at least 1, 2, 3, or 4 cousins,grandparents, parents, aunts, uncles, and/or siblings who have beendiagnosed with an inflammatory disease. In embodiments, the subject hasat least 4 cousins, grandparents, parents, aunts, uncles, and/orsiblings who have been diagnosed with an inflammatory disease. Inembodiments, the subject has at least 3 cousins, grandparents, parents,aunts, uncles, and/or siblings who have been diagnosed with aninflammatory disease. In embodiments, the subject has at least 2cousins, grandparents, parents, aunts, uncles, and/or siblings who havebeen diagnosed with an inflammatory disease. In embodiments, the subjecthas at least 1 cousin, grandparent, parent, aunt, uncle, and/or siblingwho has been diagnosed with an inflammatory disease.

In embodiments, the inflammatory disease is an allergy, atopy, asthma,an autoimmune disease, an autoinflammatory disease, a hypersensitivity,pediatric allergic asthma, allergic asthma, inflammatory bowel disease,Celiac disease, Crohn's disease, colitis, ulcerative colitis,collagenous colitis, lymphocytic colitis, diverticulitis, irritablebowel syndrome, short bowel syndrome, stagnant loop syndrome, chronicpersistent diarrhea, intractable diarrhea of infancy, Traveler'sdiarrhea, immunoproliferative small intestinal disease, chronicprostatitis, postenteritis syndrome, tropical sprue, Whipple's disease,Wolman disease, arthritis, rheumatoid arthritis, Behçet's disease,uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury,psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis,systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onsetdiabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto'sencephalitis, Hashimoto's thyroiditis, ankylosing spondylitis,psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis,auto-immune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis,Graves ophthalmopathy, Addison's disease, Vitiligo, acne vulgaris,pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplantrejection, interstitial cystitis, atherosclerosis, and atopicdermatitis.

In embodiments, the inflammatory disease is pediatric allergic asthma orinflammatory bowel disease. In embodiments, the subject suffers fromconstipation, diarrhea, bloating, urgency, and/or abdominal pain.

In embodiments, the subject has been administered an antibiotic withinthe last 1, 2, 3, or 4 months. In embodiments, the subject has beenadministered an antibiotic within the last 4 months.

In embodiments, the subject has been administered an antibiotic withinthe last 3 months. In embodiments, the subject has been administered anantibiotic within the last 2 months. In embodiments, the subject hasbeen administered an antibiotic within the last 1 month.

In embodiments, the subject is a neonate. In embodiments, the subject isless than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 18, or 24 months old. Inembodiments, the subject is less than about 1 month old. In embodiments,the subject is less than 1 month old. In embodiments, the subject isless than about 2 months old. In embodiments, the subject is less than 2months old. In embodiments, the subject is less than about 3 months old.In embodiments, the subject is less than 3 months old. In embodiments,the subject is less than about 4 months old. In embodiments, the subjectis less than 4 months old. In embodiments, the subject is less thanabout 5 months old. In embodiments, the subject is less than 5 monthsold. In embodiments, the subject is less than about 6 months old. Inembodiments, the subject is less than 6 months old. In embodiments, thesubject is less than about 7 months old. In embodiments, the subject isless than 7 months old. In embodiments, the subject is less than about 8months old. In embodiments, the subject is less than 8 months old. Inembodiments, the subject is less than about 9 months old. Inembodiments, the subject is less than 9 months old. In embodiments, thesubject is less than about 12 months old. In embodiments, the subject isless than 12 months old. In embodiments, the subject is less than about18 months old. In embodiments, the subject is less than 18 months old.In embodiments, the subject is less than about 24 months old. Inembodiments, the subject is less than 24 months old.

In embodiments, the subject is between about 2 and about 18 years old,or is at least about 18 years old. In embodiments, the subject isbetween 2 and 18 years old, or is at least 18 years old. In embodiments,the subject is between about 2 and about 18 years old, or is at leastabout 18 (e.g., 19, 20, 25, 30, 40, 50, 60, 70, 80, 90) years old. Inembodiments, the subject is between about 2 and about 18 years old, oris about 19 years old. In embodiments, the subject is between about 2and about 18 years old, or is 19 years old. In embodiments, the subjectis between about 2 and about 18 years old, or is about 20 years old. Inembodiments, the subject is between about 2 and about 18 years old, oris 20 years old. In embodiments, the subject is between about 2 andabout 18 years old, or is about 25 years old. In embodiments, thesubject is between about 2 and about 18 years old, or is 25 years old.In embodiments, the subject is between about 2 and about 18 years old,or is about 30 years old. In embodiments, the subject is between about 2and about 18 years old, or is 30 years old. In embodiments, the subjectis between about 2 and about 18 years old, or is about 40 years old. Inembodiments, the subject is between about 2 and about 18 years old, oris 40 years old. In embodiments, the subject is between about 2 andabout 18 years old, or is about 50 years old. In embodiments, thesubject is between about 2 and about 18 years old, or is 50 years old.In embodiments, the subject is between about 2 and about 18 years old,or is about 60 years old. In embodiments, the subject is between about 2and about 18 years old, or is 60 years old. In embodiments, the subjectis between about 2 and about 18 years old, or is about 70 years old. Inembodiments, the subject is between about 2 and about 18 years old, oris 70 years old. In embodiments, the subject is between about 2 andabout 18 years old, or is about 80 years old. In embodiments, thesubject is between about 2 and about 18 years old, or is 80 years old.In embodiments, the subject is between about 2 and about 18 years old,or is about 90 years old. In embodiments, the subject is between about 2and about 18 years old, or is 90 years old.

In embodiments, the subject comprises a gastrointestinal microbiome that(a) has an increased proportion of Streptococcus spp., Bifidobacteriumspp., and Enterococcus spp. compared to a healthy or general population;(b) has a reduced proportion of Alternaria alternata, Aspergillusflavus, Aspergillus cibarius, and Candida sojae compared to a healthy orgeneral population; (c) has an increased proportion of Candida albicansand Debaryomyces spp. compared to a healthy or general population; (d)has a reduced proportion of Bifdobacteria spp., Lactobacillus spp.,Faecalibacterium spp. and Akkermansia spp. compared to a healthy orgeneral population; (e) has a reduced proportion of Malassezia spp.compared to a healthy or general population; (f) has an increasedproportion of Bacterioides spp., Ruminococcus spp., Prevotella spp., orBifidobacterium spp. compared to a healthy or general population; or (g)has an increased proportion of Enterococcus faecalis, Enterococcusfaecium, or Clostridium difficile compared to a healthy or generalpopulation.

In embodiments, the effective amount is effective to (i) increase thelevel of a Bifidobacterium sp., Clostridia sp. belonging to Clade IV orXIV, a Lachnospira sp., and/or a Ruminococcus sp. in the subject; (ii)lower the pH in the feces of the subject; (iii) increase the level oflactic acid in the feces of the subject; (iv) increase the level ofcirculating itaconate in the subject; (v) treat, reduce, or preventallergic inflammation in a subject; (vi) reduce an adaptive immuneresponse in an airway of the subject; (vii) reduce dendritic cellactivation in a gastrointestinal-associated mesenteric lymoph node;(viii) increase the level of repair macrophages in the lungs, blood,serum, or plasma of the subject; (ix) increase the level of ananti-inflammatory compound in the subject; (x) decrease the level of apro-inflammatory compound in the subject; (xi) decrease the level ofeotaxin expression and/or secretion in the subject; and/or (xii)decrease the level of mucin expression and/or secretion in the subject.

In embodiments, the effective amount is effective to decrease the levelof mucin secretion and/or secretion in the lungs of the subject.

In embodiments, the anti-inflammatory compound is a cytokine, amicrobial lipid, a microbial carbohydrate, or a microbial amino acid. Inembodiments, the anti-inflammatory compound is IL-17. In embodiments,

In embodiments, the pro-inflammatory compound is a cytokine, a microbiallipid, a microbial carbohydrate, or a microbial amino acid. Inembodiments, the pro-inflammatory compound is IL-4, IL-10, IL-8, IL-13,TNF-α, or MUC5B.

In embodiments, the Lactobacillus sp., Faecalibacterium sp., Akkermansiasp., Myxococcus sp., and/or Pediococcus sp. is metabolically active. Inembodiments, the Lactobacillus sp., Faecalibacterium sp., Akkermansiasp., Myxococcus sp., and/or Pediococcus sp. is metabolically inactive.

In embodiments, the method further includes administering (a) aBifidobacterium sp., (b) Cystobacter sp., or (c) a fungal microorganismto the subject.

In embodiments, the effective amount is effective to alter themetabolism of the subject. In embodiments, altering the metabolism ofthe subject includes increasing the level of a lipid, a phospholipid, ora plasmalogen. In embodiments, altering the metabolism of the subjectincludes increasing the level of any compound listed in Table 3, or anycombination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 compounds listed in Table 3,in the subject. In embodiments, altering the metabolism of the subjectincludes increasing the level of any compound listed in Table 3, or anycombination of 2 compounds listed in Table 3, in the subject. Inembodiments, altering the metabolism of the subject includes increasingthe level of any compound listed in Table 3, or any combination of 3compounds listed in Table 3, in the subject. In embodiments, alteringthe metabolism of the subject includes increasing the level of anycompound listed in Table 3, or any combination of 4 compounds listed inTable 3, in the subject. In embodiments, altering the metabolism of thesubject includes increasing the level of any compound listed in Table 3,or any combination of 5 compounds listed in Table 3, in the subject. Inembodiments, altering the metabolism of the subject includes increasingthe level of any compound listed in Table 3, or any combination of 6compounds listed in Table 3, in the subject. In embodiments, alteringthe metabolism of the subject includes increasing the level of anycompound listed in Table 3, or any combination of 7 compounds listed inTable 3, in the subject. In embodiments, altering the metabolism of thesubject includes increasing the level of any compound listed in Table 3,or any combination of 8 compounds listed in Table 3, in the subject. Inembodiments, altering the metabolism of the subject includes increasingthe level of any compound listed in Table 3, or any combination of 9compounds listed in Table 3, in the subject. In embodiments, alteringthe metabolism of the subject includes increasing the level of anycompound listed in Table 3, or any combination of 10 compounds listed inTable 3, in the subject. In embodiments, altering the metabolism of thesubject includes increasing the level of any compound listed in Table 3,or any combination of 11 compounds listed in Table 3, in the subject. Inembodiments, altering the metabolism of the subject includes increasingthe level of any compound listed in Table 3, or any combination of 12compounds listed in Table 3, in the subject. In embodiments, alteringthe metabolism of the subject includes increasing the level of anycompound listed in Table 3, or any combination of 13 compounds listed inTable 3, in the subject. In embodiments, altering the metabolism of thesubject includes increasing the level of any compound listed in Table 3,or any combination of 14 compounds listed in Table 3, in the subject. Inembodiments, altering the metabolism of the subject includes increasingthe level of any compound listed in Table 3, or any combination of 15compounds listed in Table 3, in the subject. In embodiments, alteringthe metabolism of the subject includes increasing the level of anycompound listed in Table 3, or any combination of 16 compounds listed inTable 3, in the subject. In embodiments, altering the metabolism of thesubject includes increasing the level of any compound listed in Table 3,or any combination of 17 compounds listed in Table 3, in the subject. Inembodiments, altering the metabolism of the subject includes increasingthe level of any compound listed in Table 3, or any combination of 18compounds listed in Table 3, in the subject. In embodiments, alteringthe metabolism of the subject includes increasing the level of anycompound listed in Table 3, or any combination of 19 compounds listed inTable 3, in the subject. In embodiments, altering the metabolism of thesubject includes increasing the level of any compound listed in Table 3,or any combination of 20 compounds listed in Table 3, in the subject. Inembodiments, altering the metabolism of the subject includes increasingthe level of any compound listed in Table 3, or any combination of 21compounds listed in Table 3, in the subject. In embodiments, alteringthe metabolism of the subject includes increasing the level of anycompound listed in Table 3, or any combination of 22 compounds listed inTable 3, in the subject. In embodiments, altering the metabolism of thesubject includes increasing the level of any compound listed in Table 3,or any combination of 23 compounds listed in Table 3, in the subject. Inembodiments, altering the metabolism of the subject includes increasingthe level of any compound listed in Table 3, or any combination of 24compounds listed in Table 3, in the subject. In embodiments, alteringthe metabolism of the subject includes increasing the level of anycompound listed in Table 3, or any combination of 25 compounds listed inTable 3, in the subject. In embodiments, the level is increased in thefeces of the subject. In embodiments, the level is increased in a bodyfluid of the subject. In embodiments, altering the metabolism of thesubject comprises decreasing the level of a carbohydrate, a lipid, or anenergy compound in a subject. In embodiments, altering the metabolism ofthe subject comprises decreasing the level of any compound listed inTable 4, or any combination of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45,or 50 compounds listed in Table 4, in the subject. In embodiments,altering the metabolism of the subject includes decreasing the level ofany compound listed in Table 4, or any combination of 2 compounds listedin Table 4, in the subject. In embodiments, altering the metabolism ofthe subject includes decreasing the level of any compound listed inTable 4, or any combination of 3 compounds listed in Table 4, in thesubject. In embodiments, altering the metabolism of the subject includesdecreasing the level of any compound listed in Table 4, or anycombination of 4 compounds listed in Table 4, in the subject. Inembodiments, altering the metabolism of the subject includes decreasingthe level of any compound listed in Table 4, or any combination of 5compounds listed in Table 4, in the subject. In embodiments, alteringthe metabolism of the subject includes decreasing the level of anycompound listed in Table 4, or any combination of 6 compounds listed inTable 4, in the subject. In embodiments, altering the metabolism of thesubject includes decreasing the level of any compound listed in Table 4,or any combination of 7 compounds listed in Table 4, in the subject. Inembodiments, altering the metabolism of the subject includes decreasingthe level of any compound listed in Table 4, or any combination of 8compounds listed in Table 4, in the subject. In embodiments, alteringthe metabolism of the subject includes decreasing the level of anycompound listed in Table 4, or any combination of 9 compounds listed inTable 4, in the subject. In embodiments, altering the metabolism of thesubject includes decreasing the level of any compound listed in Table 4,or any combination of 10 compounds listed in Table 4, in the subject. Inembodiments, altering the metabolism of the subject includes decreasingthe level of any compound listed in Table 4, or any combination of 11compounds listed in Table 4, in the subject. In embodiments, alteringthe metabolism of the subject includes decreasing the level of anycompound listed in Table 4, or any combination of 12 compounds listed inTable 4, in the subject. In embodiments, altering the metabolism of thesubject includes decreasing the level of any compound listed in Table 4,or any combination of 13 compounds listed in Table 4, in the subject. Inembodiments, altering the metabolism of the subject includes decreasingthe level of any compound listed in Table 4, or any combination of 14compounds listed in Table 4, in the subject. In embodiments, alteringthe metabolism of the subject includes decreasing the level of anycompound listed in Table 4, or any combination of 15 compounds listed inTable 4, in the subject. In embodiments, altering the metabolism of thesubject includes decreasing the level of any compound listed in Table 4,or any combination of 16 compounds listed in Table 4, in the subject. Inembodiments, altering the metabolism of the subject includes decreasingthe level of any compound listed in Table 4, or any combination of 17compounds listed in Table 4, in the subject. In embodiments, alteringthe metabolism of the subject includes decreasing the level of anycompound listed in Table 4, or any combination of 18 compounds listed inTable 4, in the subject. In embodiments, altering the metabolism of thesubject includes decreasing the level of any compound listed in Table 4,or any combination of 19 compounds listed in Table 4, in the subject. Inembodiments, altering the metabolism of the subject includes decreasingthe level of any compound listed in Table 4, or any combination of 20compounds listed in Table 4, in the subject. In embodiments, alteringthe metabolism of the subject includes decreasing the level of anycompound listed in Table 4, or any combination of 21 compounds listed inTable 4, in the subject. In embodiments, altering the metabolism of thesubject includes decreasing the level of any compound listed in Table 4,or any combination of 22 compounds listed in Table 4, in the subject. Inembodiments, altering the metabolism of the subject includes decreasingthe level of any compound listed in Table 4, or any combination of 23compounds listed in Table 4, in the subject. In embodiments, alteringthe metabolism of the subject includes decreasing the level of anycompound listed in Table 4, or any combination of 24 compounds listed inTable 4, in the subject. In embodiments, altering the metabolism of thesubject includes decreasing the level of any compound listed in Table 4,or any combination of 25 compounds listed in Table 4, in the subject. Inembodiments, altering the metabolism of the subject includes decreasingthe level of any compound listed in Table 4, or any combination of 30compounds listed in Table 4, in the subject. In embodiments, alteringthe metabolism of the subject includes decreasing the level of anycompound listed in Table 4, or any combination of 35 compounds listed inTable 4, in the subject. In embodiments, altering the metabolism of thesubject includes decreasing the level of any compound listed in Table 4,or any combination of 40 compounds listed in Table 4, in the subject. Inembodiments, altering the metabolism of the subject includes decreasingthe level of any compound listed in Table 4, or any combination of 45compounds listed in Table 4, in the subject. In embodiments, alteringthe metabolism of the subject includes decreasing the level of anycompound listed in Table 4, or any combination of 50 compounds listed inTable 4, in the subject. In embodiments, the level is decreased in thefeces of the subject. In embodiments, the level is decreased in a bodyfluid of the subject.

In an aspect is provided a method of treating or preventing aninflammatory disease in a subject in need thereof. The method includesadministering to the subject a therapeutically effective amount ofLactobacillus johnsonii. Faecalibacterium prausnitzii. Akkermansiamuciniphila, Myxococcus xanthus and Pediococcus pentosaceus. Inembodiments, the Lactobacillus johnsonii, Faecalibacterium prausnitzii,Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceusform a microbial composition as provided herein. Where the Lactobacillusjohnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila,Myxococcus xanthus and Pediococcus pentosaceus form a microbialcomposition, the bacteria form part of a composition including apharmaceutically acceptable carrier for administration to andcolonialization of the gut. Acceptable carriers include, but are notlimited to inulin. In embodiments, the gut is of a healthy subject. Inembodiments, the gut is of a subject in need of treatment or preventionof an inflammatory disease. In embodiments, the subject is a neonate. A“neonate” as provided herein refers to a newborn child or mammal. Inembodiments, the neonate is less than about four weeks old.

In an aspect, a method of treating or preventing an inflammatory diseasein a subject in need thereof is provided. The method includingadministering to the subject an effective amount of a bacterialpopulation comprising Lactobacillus sp., Faecalibacterium sp.,Akkermansia sp., Myxococcus sp., and Pediococcus sp.

In embodiments, the inflammatory disease is an allergy, atopy, asthma,an autoimmune disease, an autoinflammatory disease, a hypersensitivity,pediatric allergic asthma, allergic asthma, inflammatory bowel disease,Celiac disease, Crohn's disease, colitis, ulcerative colitis,collagenous colitis, lymphocytic colitis, diverticulitis, irritablebowel syndrome, short bowel syndrome, stagnant loop syndrome, chronicpersistent diarrhea, intractable diarrhea of infancy, Traveler'sdiarrhea, immunoproliferative small intestinal disease, chronicprostatitis, postenteritis syndrome, tropical sprue, Whipple's disease,Wolman disease, arthritis, rheumatoid arthritis, Behçet's disease,uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury,psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis,systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onsetdiabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto'sencephalitis, Hashimoto's thyroiditis, ankylosing spondylitis,psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis,auto-immune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis,Graves ophthalmopathy, Addison's disease, Vitiligo, acne vulgaris,pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplantrejection, interstitial cystitis, atherosclerosis, and atopicdermatitis.

In embodiments, the Lactobacillus johnsonii, Faecalibacteriumprausnitzii, Akkermansia muciniphila, Myxococcus xanthus and Pediococcuspentosaceus are metabolically active. “Metabolically active” as providedherein refer to cells (e.g., bacteria) capable of cell division. Inembodiments the metabolically active cell is capable of substrate (e.g.glucose) consumption. In embodiments, the Lactobacillus johnsonii,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus and Pediococcus pentosaceus are metabolically inactive. Inembodiments, the microbial composition is effective for administrationto the gut. In embodiments, the microbial composition does not includeLactobacillus rhamnosus.

In embodiments, the microbial composition is effective to increase ananti-inflammatory metabolite (e.g., microbial lipid, a microbialcarbohydrate or a microbial amino acid). As described herein, theanti-inflammatory metabolite may be a microbial lipid (e.g.,phospholipid, poly-unsaturated fatty acid). In embodiments, theanti-inflammatory metabolite is a phospholipid. In embodiments, theanti-inflammatory metabolite is poly-unsaturated fatty acid. Inembodiments, the anti-inflammatory metabolite is a microbialcarbohydrate (e.g., itoconate, n-acetylglucosamine,n-acetylgalactosamine, fucosyllactose). In embodiments, theanti-inflammatory metabolite is itoconate. In embodiments, theanti-inflammatory metabolite is n-acetylglucosamine. In embodiments, theanti-inflammatory metabolite is n-acetylgalactosamine. In embodiments,the anti-inflammatory metabolite is fucosyllactose. In embodiments, theanti-inflammatory metabolite is a microbial amino acid (e.g.,tryptophan). In embodiments, the anti-inflammatory metabolite istryptophan. A composition capable of increasing an anti-inflammatorymetabolite as provided herein refers to a composition that results in adetectably higher level of an anti-inflammatory metabolite as comparedto a control. The increased activity can be 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90% or more than that in a control. In certain instances,the increase is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, ormore in comparison to a control. In embodiments, the microbialcomposition is effective to increase the number of IL-17-secreting Thelper cells.

In embodiments, the microbial composition is effective to decrease apro-inflammatory metabolite. As described herein, the pro-inflammatorymetabolite may be a microbial lipid (e.g., dihydroxyoctadec-12-enoicacid, cholate or methylmalonate). In embodiments, the pro-inflammatorymetabolite is dihydroxyoctadec-12-enoic acid. In embodiments, thepro-inflammatory metabolite is a cholate. In embodiments, thepro-inflammatory metabolite is methylmalonate. In embodiments, thepro-inflammatory metabolite is a microbial carbohydrate (e.g.,n-acetylymuramate, lactobionate or maltotriose). In embodiments, thepro-inflammatory metabolite is n-acetylymuramate. In embodiments, thepro-inflammatory metabolite is lactobionate. In embodiments, thepro-inflammatory metabolite is maltotriose. In embodiments, thepro-inflammatory metabolite is a microbial amino acid (e.g., ornithineor taurine). In embodiments, the pro-inflammatory metabolite isornithine. In embodiments, the pro-inflammatory metabolite is taurine. Acomposition capable of decreasing a pro-inflammatory metabolite asprovided herein refers to a composition that results in a detectablylower level of a pro-inflammatory metabolite as compared to a control.The decreased activity can be 10%, 20%, 300, 40%, 50%, 60%, 70%, 80%,90% or less than that in a control. In certain instances, the decreaseis 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or less incomparison to a control.

In embodiments, the pro-inflammatory metabolite is IL-4, IL-10, IL-13 orMUC5B. In embodiments, the pro-inflammatory metabolite is IL-4. Inembodiments, the pro-inflammatory metabolite is IL-10. In embodiments,the pro-inflammatory metabolite is IL-13. In embodiments, thepro-inflammatory metabolite MUC5B. In embodiments, the pro-inflammatorymetabolite MUC5AC. In embodiments, the microbial composition iseffective to decrease T helper cell type 2 cytokine expression.

The term “IL-4” as provided herein includes any of the recombinant ornaturally-occurring forms of the interleukin 4 (IL-4) cytokine orvariants or homologs thereof that maintain IL-4 protein activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to IL-4). In some aspects, the variants or homologs have atleast 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityacross the whole sequence or a portion of the sequence (e.g. a 50, 100,150 or 200 continuous amino acid portion) compared to a naturallyoccurring IL-4 polypeptide. In embodiments, 1-4 is the protein asidentified by the NCBI sequence reference GI:4504669 (Accession No.NP_000580.1; SEQ ID NO: 1), or an isoform, a homolog or functionalfragment thereof.

The term “IL-10” as provided herein includes any of the recombinant ornaturally-occurring forms of the interleukin 10 (IL-10) cytokine orvariants or homologs thereof that maintain IL-10 protein activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to IL-10). In some aspects, the variants or homologs have atleast 90%, 95%, 96%, 97%, 98%, 99⁰% or 100% amino acid sequence identityacross the whole sequence or a portion of the sequence (e.g. a 50, 100,150 or 200 continuous amino acid portion) compared to a naturallyoccurring IL-10 polypeptide. In embodiments, IL-10 is the protein asidentified by the NCBI sequence reference GI:10835141 (Accession No.NP_000563.1; SEQ ID NO:2), or an isoform, a homolog or functionalfragment thereof.

The term “IL-13” as provided herein includes any of the recombinant ornaturally-occurring forms of the interleukin 13 (IL-13) cytokine orvariants or homologs thereof that maintain IL-13 protein activity (e.g.within at least 50%, 80%, 90%, 95%, 96° %, 97%, 98%, 99% or 100%activity compared to IL-13). In some aspects, the variants or homologshave at least 90° %, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring IL-13 polypeptide. In embodiments, IL-13 is theprotein as identified by the NCBI sequence reference GI:26787978(Accession No. NP_002179.2; SEQ ID NO:3), or an isoform, a homolog orfunctional fragment thereof.

The term “IL-17” as provided herein includes any of the recombinant ornaturally-occurring forms of the interleukin 17 (IL-17) cytokine orvariants or homologs thereof that maintain IL-17 protein activity (e.g.within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to IL-17). In some aspects, the variants or homologs have atleast 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identityacross the whole sequence or a portion of the sequence (e.g. a 50, 100,150 or 200 continuous amino acid portion) compared to a naturallyoccurring IL-17 polypeptide. In embodiments, IL-17 is the protein asidentified by the UniProt sequence reference Q16552 (SEQ ID NO:4), or ahomolog or functional fragment thereof. In embodiments, IL-17 is theprotein as identified by the UniProt sequence reference Q9UHF5, or anisoform, a homolog or functional fragment thereof.

The term “MUC5AC” as provided herein includes any of the recombinant ornaturally-occurring forms of the mucin 5AC (MUC5AC) protein or variantsor homologs thereof that maintain MUC5AC protein activity (e.g. withinat least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activitycompared to MUC5AC). In some aspects, the variants or homologs have atleast 90%, 95%, 96%, 97%, 98%, 99% or 100%0, amino acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a50, 100, 150 or 200 continuous amino acid portion) compared to anaturally occurring MUC5AC polypeptide. In embodiments, MUC5AC is theprotein as identified by the UniProt sequence reference P98088 (SEQ IDNO:5), or an isoform, a homolog or functional fragment thereof.

The term “MUC5B” as provided herein includes any of the recombinant ornaturally-occurring forms of the mucin 5B (MUC5B) protein or variants orhomologs thereof that maintain MUC5B protein activity (e.g. within atleast 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%0/activity comparedto MUC5B). In some aspects, the variants or homologs have at least 90%,95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity across thewhole sequence or a portion of the sequence (e.g. a 50, 100, 150 or 200continuous amino acid portion) compared to a naturally occurring MUC5Bpolypeptide. In embodiments, MUC5B is the protein as identified by theUniProt sequence reference Q9HC84 (SEQ ID NO:6), or an isoform, ahomolog or functional fragment thereof.

In embodiments, the method further includes administering atherapeutically effective amount of a fungus. In embodiments, the fungusis a Malassezia fungus. In embodiments, the microbial composition iseffective to decrease a pathogenic fungal activity. A “pathogenic fungalactivity” as referred to herein is a metabolic activity derived from apathogenic fungus. In embodiments, the pathogenic fungus is Candidaalbicans. In embodiments, the pathogenic fungus forms a pro-inflammatorylipid.

In embodiments, the subject is a neonate. In embodiments, the neonate isless than about four weeks old. In embodiments, the neonate is treatedfor at least about one month. In embodiments, the neonate is treated forat least about two months. In embodiments, the neonate is treated for atleast about three months. In embodiments, the neonate is treated for atleast about four months. In embodiments, the neonate is treated for atleast about five months. In embodiments, the neonate is treated for atleast about six months.

In embodiments, the neonate is treated for about one month. Inembodiments, the neonate is treated for about two months. Inembodiments, the neonate is treated for about three months. Inembodiments, the neonate is treated for about four months. Inembodiments, the neonate is treated for about five months. Inembodiments, the neonate is treated for about six months.

In embodiments, the neonate is treated for less than about one month. Inembodiments, the neonate is treated for less than about two months. Inembodiments, the neonate is treated for less than about three months. Inembodiments, the neonate is treated for less than about four months. Inembodiments, the neonate is treated for less than about five months. Inembodiments, the neonate is treated for less than about six months.

In embodiments, the neonate is treated for about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 51, 52, 53, or 54, weeks.

In embodiments, the inflammatory disease is asthma, ulcerative colitis,irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum orerythema nodosum. In embodiments, the inflammatory disease is asthma. Inembodiments, the inflammatory disease is ulcerative colitis. Inembodiments, the inflammatory disease is irritable bowel syndrome. Inembodiments, the inflammatory disease is arthritis. In embodiments, theinflammatory disease is uveitis. In embodiments, the inflammatorydisease is pyoderma gangrenosum. In embodiments, the inflammatorydisease is erythema nodosum.

In an aspect, a method of increasing an anti-inflammatory metabolite ina subject in need thereof is provided. The method includes administeringto the subject a therapeutically effective amount of Lactobacillusjohnsonii, Faecalibacterium prausnitzii. Akkermansia muciniphila,Myxococcus xanthus and Pediococcus pentosaceus. In embodiments, themethod further includes a pharmaceutically active excipient as providedherein. The Lactobacillus johnsonii, Faecalibacterium prausnitzii,Akkermansia muciniphila, Myxococcus xanthus and Pediococcus pentosaceusmay form a microbial composition provided herein including embodimentsthereof. Thus, in embodiments, the Lactobacillus johnsonii,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus and Pediococcus pentosaceus form a microbial composition.

The anti-inflammatory metabolite may be a microbial lipid (e.g., aphospholipid, a poly unsaturated fatty acid), a microbial carbohydrate(e.g., itoconate, n-acetylglucosamine, n-acetylgalactosamine,fucosyllactose) or a microbial amino acid (e.g., tryptophan) as providedherein. In embodiments, the anti-inflammatory metabolite is a microbiallipid. In embodiments, the anti-inflammatory metabolite is a microbialcarbohydrate. In embodiments, the anti-inflammatory metabolite is amicrobial amino acid.

In an aspect is provided a method of decreasing a pro-inflammatorymetabolite in a subject in need thereof. The method includesadministering to the subject a therapeutically effective amount ofLactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansiamuciniphila, Myxococcus xanthus and Pediococcus pentosaceus. Inembodiments, the method further includes a pharmaceutically activeexcipient as provided herein. The Lactobacillus johnsonii,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus and Pediococcus pentosaceus may form a microbial compositionprovided herein including embodiments thereof. Thus, in embodiments, theLactobacillus johnsonii, Faecalibacterium prausnitzii, Akkermansiamuciniphila, Myxococcus xanthus and Pediococcus pentosaceus form amicrobial composition.

As described herein, the pro-inflammatory metabolite may be a microbiallipid (e.g., dihydroxyoctadec-12-enoic acid, cholate or methylmalonate).In embodiments, the pro-inflammatory metabolite isdihydroxyoctadec-12-enoic acid. In embodiments, the pro-inflammatorymetabolite is a cholate. In embodiments, the pro-inflammatory metaboliteis methylmalonate. In embodiments, the pro-inflammatory metabolite is amicrobial carbohydrate (e.g., n-acetylymuramate, lactobionate ormaltotriose). In embodiments, the pro-inflammatory metabolite isn-acetylymuramate. In embodiments, the pro-inflammatory metabolite islactobionate. In embodiments, the pro-inflammatory metabolite ismaltotriose. In embodiments, the pro-inflammatory metabolite is amicrobial amino acid (e.g., ornithine or taurine). In embodiments, thepro-inflammatory metabolite is ornithine. In embodiments, thepro-inflammatory metabolite is taurine.

In an aspect, a method of increasing the level of an anti-inflammatorycompound and/or decreasing the level of a pro-inflammatory compound in asubject in need thereof is provided. In embodiments, the method includesadministering to the subject an effective amount of a bacterialpopulation comprising Lactobacillus sp., Faecalibacterium sp.,Akkermansia sp., Myxococcus sp., and Pediococcus sp.

In embodiments, the method for increasing the level of theanti-inflammatory compound increases and/or decreases the level of thepro-inflammatory compound in the feces, blood, plasma, serum,broncheoalveolar lavage fluid, sweat, saliva, sputum, lymph, spinalfluid, urine, tears, bile, aqueous humour, vitreous humour, aminioticfluid, breast milk, cerebrospinal fluid, cerumen, nasal mucus, phlegm,or sebum of the subject.

In embodiments, the anti-inflammatory compound is a microbial lipid, amicrobial carbohydrate, or a microbial amino acid.

In embodiments, the subject suffers from dysbiosis or an inflammatorydisease.

In embodiments, the inflammatory disease is a disease as describedherein. In embodiments, the inflammatory disease is ulcerative colitis.In embodiments, the inflammatory disease is irritable bowel syndrome. Inembodiments, the inflammatory disease is arthritis. In embodiments, theinflammatory disease is uveitis. In embodiments, the inflammatorydisease is pyoderma gangrenosum. In embodiments, the inflammatorydisease is erythema nodosum.

In an aspect, a method of treating or preventing a viral respiratoryinfection in a subject in need thereof is provided. The method includingadministering to the subject an effective amount of a bacterialpopulation comprising Lactobacillus sp., Faecalibacterium sp.,Akkermansia sp., Myxococcus sp., and Pediococcus sp.

In embodiments, wherein the viral respiratory infection is caused by arespiratory syncytial virus, an influenza virus, a parainfluenza virus,an adenovirus, a coronavirus, or a rhinovirus. In embodiments, the viralrespiratory infection is bronchiolitis, a cold, croup, or pneumonia.

In aspects is provided a method of treating or preventing an allergy ina subject in need thereof. The method including administering to thesubject an effective amount of a bacteria population comprisingLactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcussp., and Pediococcus sp.

In embodiments, the allergy is an allergy to milk, eggs, fish,shellfish, a tree nut, peanuts, wheat, dander from a cat, dog, orrodent, an insect sting, pollen, latex, dust mites, or soybeans. Inembodiments, the allergy is pediatric allergic asthma, hay fever, orallergic airway sensitization.

V. Methods of Detection

In an aspect a method of detecting an anti-inflammatory metabolite in asubject that has or is at risk for developing an inflammatory disease isprovided. The method includes (i) obtaining a biological sample from thesubject; and (ii) determining an expression level of ananti-inflammatory metabolite in the biological sample. Theanti-inflammatory metabolite may be a microbial lipid (e.g., aphospholipid, a poly unsaturated fatty acid), a microbial carbohydrate(e.g., itoconate, n-acetylglucosamine, n-acetylgalactosamine,fucosyllactose) or a microbial amino acid (e.g., tryptophan) as providedherein. Thus, in embodiments, the anti-inflammatory metabolite is amicrobial lipid or a microbial carbohydrate as described herein. Inembodiments, the anti-inflammatory metabolite is a microbial lipid. Inembodiments, the anti-inflammatory metabolite is a microbialcarbohydrate.

In embodiments, the expression level of a compound (e.g., a metabolite)is the amount (e.g., weight) of the compound. In embodiments, theexpression level of a compound (e.g., a protein such as a cytokine) isthe level of mRNA expression.

In an aspect a method of detecting a pro-inflammatory metabolite in asubject that has or is at risk for developing an inflammatory disease isprovided. The method includes (i) obtaining a biological sample from thesubject; and (ii) determining an expression level of a pro-inflammatorymetabolite in the biological sample. The pro-inflammatory metabolite maybe a microbial lipid (e.g., dihydroxyoctadec-12-enoic acid, cholate ormethylmalonate), a microbial carbohydrate (e.g., n-acetylymuramate,lactobionate or maltotriose syllactose) or a microbial amino acid (e.g.,ornithine or taurine) as provided herein.

In an aspect, a method of detecting a pro-inflammatory compound in asubject in need thereof is provided. In embodiments, the method includes(i) obtaining a biological sample from the subject; and (ii) detectingthe pro-inflammatory compound in the biological sample.

In an aspect, a method of monitoring the effect of treatment fordysbiosis or an inflammatory disease is provided. In embodiments, themethod includes (i) obtaining a biological sample from the subject; and(ii) detecting whether the biological sample is pro-inflammatory.

In an aspect, a method of determining an inflammatory disease activityin a subject is provided. In embodiments, the method includes (i)obtaining a biological sample from the subject; and (ii) detectingwhether the biological sample is pro-inflammatory.

In an aspect, a method of detecting an anti-inflammatory metabolite in asubject that has or is at risk for developing an inflammatory disease isprovided. In embodiments, the method includes (i) obtaining a biologicalsample from the subject; and (ii) determining an expression level of ananti-inflammatory metabolite in the biological sample.

In embodiments, the subject has or is at risk for developing dysbiosis.In embodiments, the subject has an inflammatory disease. In embodiments,the subject is at risk of suffering from an inflammatory disease.

In embodiments, the subject (i) has at least 1, 2, 3, or 4 cousins,grandparents, parents, aunts, uncles, and/or siblings who have beendiagnosed with an inflammatory disease; (ii) suffers from constipation,diarrhea, bloating, urgency, and/or abdominal pain; and/or (iii) hasbeen administered an antibiotic within the last 1, 2, or 4 months.

In embodiments, the inflammatory disease is an allergy, atopy, asthma,an autoimmune disease, an autoinflammatory disease, a hypersensitivity,pediatric allergic asthma, allergic asthma, inflammatory bowel disease,Celiac disease, Crohn's disease, colitis, ulcerative colitis,collagenous colitis, lymphocytic colitis, diverticulitis, irritablebowel syndrome, short bowel syndrome, stagnant loop syndrome, chronicpersistent diarrhea, intractable diarrhea of infancy, Traveler'sdiarrhea, immunoproliferative small intestinal disease, chronicprostatitis, postenteritis syndrome, tropical sprue, Whipple's disease,Wolman disease, arthritis, rheumatoid arthritis, Behçet's disease,uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury,psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis,systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onsetdiabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto'sencephalitis, Hashimoto's thyroiditis, ankylosing spondylitis,psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis,auto-immune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis,Graves ophthalmopathy, Addison's disease, Vitiligo, acne vulgaris,pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplantrejection, interstitial cystitis, atherosclerosis, and atopicdermatitis.

In embodiments, the subject is less than about 1, 2, 3, 4, 5, 6, 7, 8,9, 12, 18, or 24 months old. In embodiments, the subject is less thanabout 1 month old. In embodiments, the subject is less than 1 month old.In embodiments, the subject is less than about 2 months old. Inembodiments, the subject is less than 2 months old. In embodiments, thesubject is less than about 3 months old. In embodiments, the subject isless than 3 months old. In embodiments, the subject is less than about 4months old. In embodiments, the subject is less than 4 months old. Inembodiments, the subject is less than about 5 months old. Inembodiments, the subject is less than 5 months old. In embodiments, thesubject is less than about 6 months old. In embodiments, the subject isless than 6 months old. In embodiments, the subject is less than about 7months old. In embodiments, the subject is less than 7 months old. Inembodiments, the subject is less than about 8 months old. Inembodiments, the subject is less than 8 months old. In embodiments, thesubject is less than about 9 months old. In embodiments, the subject isless than 9 months old. In embodiments, the subject is less than about12 months old. In embodiments, the subject is less than 12 months old.In embodiments, the subject is less than about 18 months old. Inembodiments, the subject is less than 18 months old. In embodiments, thesubject is less than about 24 months old. In embodiments, the subject isless than 24 months old.

In embodiments, the subject is between about 2 and about 18 years old,or is at least about 18 years old. In embodiments, the subject isbetween 2 and 18 years old, or is at least 18 years old. In embodiments,the subject is between about 2 and about 18 years old, or is at leastabout 18 (e.g., 19, 20, 25, 30, 40, 50, 60, 70, 80, 90) years old. Inembodiments, the subject is between about 2 and about 18 years old, oris about 19 years old. In embodiments, the subject is between about 2and about 18 years old, or is 19 years old. In embodiments, the subjectis between about 2 and about 18 years old, or is about 20 years old. Inembodiments, the subject is between about 2 and about 18 years old, oris 20 years old. In embodiments, the subject is between about 2 andabout 18 years old, or is about 25 years old. In embodiments, thesubject is between about 2 and about 18 years old, or is 25 years old.In embodiments, the subject is between about 2 and about 18 years old,or is about 30 years old. In embodiments, the subject is between about 2and about 18 years old, or is 30 years old. In embodiments, the subjectis between about 2 and about 18 years old, or is about 40 years old. Inembodiments, the subject is between about 2 and about 18 years old, oris 40 years old. In embodiments, the subject is between about 2 andabout 18 years old, or is about 50 years old. In embodiments, thesubject is between about 2 and about 18 years old, or is 50 years old.In embodiments, the subject is between about 2 and about 18 years old,or is about 60 years old. In embodiments, the subject is between about 2and about 18 years old, or is 60 years old. In embodiments, the subjectis between about 2 and about 18 years old, or is about 70 years old. Inembodiments, the subject is between about 2 and about 18 years old, oris 70 years old. In embodiments, the subject is between about 2 andabout 18 years old, or is about 80 years old. In embodiments, thesubject is between about 2 and about 18 years old, or is 80 years old.In embodiments, the subject is between about 2 and about 18 years old,or is about 90 years old. In embodiments, the subject is between about 2and about 18 years old, or is 90 years old.

In embodiments, the subject comprises a gastrointestinal microbiome that(a) has an increased proportion of Streptococcus spp., Bifidobacteriumspp., and Enterococcus spp. compared to a healthy or general population;(b) has a reduced proportion of Alternaria alternata, Aspergillusflavus, Aspergillus cibarius, and Candida sojae compared to a healthy orgeneral population; (c) has an increased proportion of Candida albicansand Debaryomyces spp. compared to a healthy or general population; (d)has a reduced proportion of Bifidobacteria spp., Lactobacillus spp.,Faecalibacterium spp. and Akkermansia spp. compared to a healthy orgeneral population; (e) has a reduced proportion of Malassezia spp.compared to a healthy or general population; (f) has an increasedproportion of Bacterioides spp., Ruminococcus spp., Prevotella spp., orBifidobacterium spp. compared to a healthy or general population; or (g)has an increased proportion of Enterococcus faecalis, Enterococcusfaecium, or Clostridium difficile compared to a healthy or generalpopulation.

In embodiments, the biological sample is a bodily fluid. In embodiments,wherein the bodily fluid is blood, plasma, serum, fecal water, or abrancheoaleolar lavage. In embodiments, the bodily fluid is fecal water.

In embodiments, detecting the pro-inflammatory compound includescontacting an antigen presenting cell with the biological sample. Inembodiments, the antigen presenting cell is a dendritic cell. Inembodiments, the dendritic cell has been isolated from blood. Inembodiments, the dendritic cell has been isolated from the blood of ahealthy subject (e.g., a subject who does not have an inflammatorydisease, an infection, and who has not been administered an antibioticwithin about 1, 2, 3, 4, 5, or 6 months). In embodiments, the dendriticcell has been obtained (e.g., isolated, selected, or enriched) fromperipheral blood mononuclear cells. In embodiments, the dendritic cellis part of a primary culture of dendritic cells. In embodiments, thedendritic cell is part of a culture of dendritic cells that has beenpassaged less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. Inembodiments, the dendritic cell is not immortalized. In embodiments, thedendritic cell is an immortalized dendritic cell.

In embodiments, detecting the pro-inflammatory compound further includescontacting a naïve T cell with the antigen presenting cell to produce acontacted T cell. In embodiments, the method further includes detectinga cytokine produced by the contacted T cell and/or the progeny of thecontacted T cell. In embodiments, the T cell has been isolated fromblood. In embodiments, the T cell has been isolated from the blood of ahealthy subject (e.g., a subject who does not have an inflammatorydisease, an infection, and who has not been administered an antibioticwithin about 1, 2, 3, 4, 5, or 6 months). In embodiments, the T cell hasbeen obtained (e.g., isolated, selected, or enriched) from peripheralblood mononuclear cells. In embodiments, the T cell is part of a primaryculture of T cells. In embodiments, the T cell is part of a culture of Tcells that has been passaged less than about 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 times. In embodiments, the T cell is not immortalized. Inembodiments, the T cell is an immortalized T cell.

In embodiments, the pro-inflammatory compound is detected if (i) theproportion of T-helper (TH)-2 cells is increased in the progeny of thecontacted T cell compared to a control; (ii) the proportion of TH-1,TH-17, and/or TH22 cells is increased in the progeny of the contacted Tcell compared to a control; (iii) the ratio of TH-1 cells to TH-2 cellsis decreased in the progeny of the contacted T cell compared to acontrol; (iv) the proportion of IL-17 producing CD8+ T cells isincreased in the progeny of the contacted T cell compared to a control;and/or (v) the amount of IL-4, IL-10, and/or IL-13 produced by theprogeny of the contacted T cell and/or the progeny thereof is increasedcompared to a control.

In embodiments, the control is (i) the corresponding proportion, ratio,and/or amount of a corresponding T cell that has been contacted withsterile culture medium and/or the progeny thereof; (ii) thecorresponding proportion, ratio, and/or amount of a corresponding T cellthat has been contacted with an antigen presenting cell that has beencontacted with a biological sample from a subject who does not havedysbiosis, an inflammatory disease, or a gastrointestinal infection,and/or the progeny thereof; and/or (iii) a reference value correspondingto the proportion, ratio, and/or amount in the general population or apopulation of subjects who do not have dysbiosis, an inflammatorydisease, or a gastrointestinal infection.

In embodiments, the method further includes directing the subject toreceive treatment or further testing or monitoring for dysbiosis or aninflammatory disease if the pro-inflammatory compound is detected in thesubject.

In embodiments, the method further includes administering thecomposition as described herein including embodiments thereof to thesubject if the pro-inflammatory compound is detected in the subject.

In embodiments, the method further includes diagnosing the subject ashaving or at risk of developing dysbiosis or an inflammatory disease ifthe pro-inflammatory compound is detected in the subject.

In embodiments, a method of determining whether a subject has or is atrisk of developing dysbiosis or an inflammatory disease is provided. Inembodiments, the method includes (i) obtaining a biological sample fromthe subject; and (ii) detecting a pro-inflammatory compound according toa method described herein including embodiments thereof.

In some examples of the disclosed methods, when the expression level ofan anti-inflammatory metabolite or pro-inflammatory metabolite isassessed, the level is compared with a control expression level of theanti-inflammatory metabolite or pro-inflammatory metabolite. By controllevel is meant the expression level of a particular an anti-inflammatorymetabolite or pro-inflammatory metabolite from a sample or subjectlacking a disease (e.g. an inflammatory disease), at a selected stage ofa disease or disease state, or in the absence of a particular variablesuch as a therapeutic agent. Alternatively, the control level comprisesa known amount of anti-inflammatory metabolite or pro-inflammatorymetabolite. Such a known amount correlates with an average level ofsubjects lacking a disease, at a selected stage of a disease or diseasestate, or in the absence of a particular variable such as a therapeuticagent. A control level also includes the expression level of one or moreanti-inflammatory metabolites or pro-inflammatory metabolites from oneor more selected samples or subjects as described herein. For example, acontrol level includes an assessment of the expression level of one ormore anti-inflammatory metabolites or pro-inflammatory metabolites in asample from a subject that does not have a disease (e.g. an inflammatorydisease), is at a selected stage of progression of a disease (e.g.inflammatory disease), or has not received treatment for a disease.Another exemplary control level includes an assessment of the expressionlevel of one or more anti-inflammatory metabolites or pro-inflammatorymetabolite in samples taken from multiple subjects that do not have adisease, are at a selected stage of progression of a disease, or havenot received treatment for a disease.

When the control level includes the expression level of one or moreanti-inflammatory metabolites or pro-inflammatory metabolites in asample or subject in the absence of a therapeutic agent (e.g., themicrobial composition provided herein including embodiments thereof),the control sample or subject is optionally the same sample or subjectto be tested before or after treatment with a therapeutic agent or is aselected sample or subject in the absence of the therapeutic agent.Alternatively, a control level is an average expression level calculatedfrom a number of subjects without a particular disease. A control levelalso includes a known control level or value known in the art.

In embodiments, the biological sample is a bodily fluid. In embodiments,the bodily fluid is serum, fecal water or brancheoaleolar lavage. Inembodiments, the bodily fluid is serum. In embodiments, the bodily fluidis fecal water. In embodiments, the bodily fluid is brancheoaleolarlavage. In embodiments, the biological sample is a tissue. Inembodiments, the tissue is lung, spleen, or ileum tissue. Inembodiments, the biological sample is a cell. In embodiments, thebiological sample is a lung cell. In embodiments, the biological sampleis a spleen cell. In embodiments, the biological sample is an ileumcell. In embodiments, the sample includes one or more bacterial cells.

In embodiments, a biological sample is a bodily fluid obtained byfiltration and/or centrifugation. For example, the biological sample maybe a filtrate of e.g., blood or feces or the supernatant of centrifugedblood or feces. In embodiments, a filtrate is centrifuged. Inembodiments a supernatant is filtered. In embodiments, centrifugation isused to increase the passage of a fluid through a filter. Non-limitingexamples of filters include filters that restrict any molecule greaterthan, e.g., 50, 100, 200, 300, 400, 500, 50-500, 50-100, 100-500 nm indiameter (or average diameter), or greater than 0.5, 1, 1.5, 2, 2.5, 5,10, 15, 25, 50, 100, or 200 microns in diameter (e.g., averagediameter). In embodiments, a filter has pores of about 50, 100, 200,300, 400, 500, 50-500, 50-100, 100-500 nm in diameter or about 0.5, 1,1.5, 2, 2.5, 5, 10, 15, 25, 50, 100, or 200 microns in diameter.

In embodiments, detecting a compound (e.g., a metabolite) and/or theexpression level thereof comprises High performance liquidchromatography (HPLC), gas chromatography, liquid chromatography, Massspectrometry (MS), inductively coupled plasma-mass spectrometry(ICP-MS), accelerator mass spectrometry (AMS), thermal ionization-massspectrometry (TIMS) and spark source mass spectrometry (SSMS),matrix-assisted laser desorption/ionization (MALDI), and/or MALDI-TOF.

In embodiments, detecting the expression level of a compound compriseslysing a cell. In embodiments, detecting the expression level of acompound comprises a polymerase chain reaction (e.g., reversetranscriptase polymerase chain reaction), microarray analysis,immunohistochemistry, or flow cytometry.

In embodiments, the determining includes: (a) contacting in vitro theanti-inflammatory metabolite with an antigen presenting cell, therebyforming a metabolite-antigen presenting cell; (b) contacting themetabolite-antigen presenting cell with a T cell, thereby forming acontacted T cell; and (c) detecting a cytokine produced by the contactedT cell. In embodiments, the cytokine is produced by an activated ordifferentiating T cell.

In embodiments, the determining includes: (a) contacting in vitro thepro-inflammatory metabolite with an antigen presenting cell, therebyforming a metabolite-antigen presenting cell; (b) contacting themetabolite-antigen presenting cell with a T cell, thereby forming acontacted T cell; (c) detecting a cytokine produced by the contacted Tcell.

In embodiments, the inflammatory disease is asthma, ulcerative colitis,irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, orerythema nodosum. In embodiments, the inflammatory disease is asthma. Inembodiments, the inflammatory disease is ulcerative colitis. Inembodiments, the inflammatory disease is irritable bowel syndrome. Inembodiments, the inflammatory disease is arthritis. In embodiments, theinflammatory disease is uveitis. In embodiments, the inflammatorydisease is pyoderma gangrenosum. In embodiments, the inflammatorydisease is erythema nodosum.

In an aspect, a method of determining whether a subject has or is atrisk of developing an inflammatory disease is provided. The methodincludes (i) detecting an expression level of one or moreanti-inflammatory metabolites or pro-inflammatory metabolites in asubject; (ii) determining whether the expression level is increased ordecreased relative to a standard control, wherein an elevated expressionlevel of an pro-inflammatory metabolite or a decreased expression levelof an anti-inflammatory metabolite relative to the standard controlindicates that the subject has or is at risk of developing aninflammatory disease; and (iii) based at least in part on the expressionlevel in step (ii), determining whether the subject has or is at riskfor developing an inflammatory disease.

In an aspect, a method of determining whether a subject has or is atrisk of developing an inflammatory disease is provided. The methodincludes (i) detecting an expression level of one or morepro-inflammatory metabolites in a subject; (ii) determining whether theexpression level is increased or decreased relative to a standardcontrol, wherein an increased expression level of an pro-inflammatorymetabolite relative to the standard control indicates that the subjecthas or is at risk of developing an inflammatory disease; and (iii) basedat least in part on the expression level in step (ii), determiningwhether the subject has or is at risk for developing an inflammatorydisease.

In another aspect, a method of determining whether a subject has or isat risk of developing an inflammatory disease is provided. The methodincludes (i) detecting an expression level of one or moreanti-inflammatory metabolites in a subject; (ii) determining whether theexpression level is increased or decreased relative to a standardcontrol, wherein a decreased expression level of an anti-inflammatorymetabolite relative to the standard control indicates that the subjecthas or is at risk of developing an inflammatory disease; and (iii) basedat least in part on the expression level in step (ii), determiningwhether the subject has or is at risk for developing an inflammatorydisease.

In embodiments, the anti-inflammatory metabolite is a microbial lipid, amicrobial carbohydrate or a microbial amino acid as provided herein. Theanti-inflammatory metabolite may be a microbial lipid (e.g., aphospholipid, a poly unsaturated fatty acid), a microbial carbohydrate(e.g., itoconate, n-acetylglucosamine, n-acetylgalactosamine,fucosyllactose) or a microbial amino acid (e.g., tryptophan) as providedherein. In embodiments, the anti-inflammatory metabolite is a microbiallipid. In embodiments, the anti-inflammatory metabolite is a microbialcarbohydrate. In embodiments, the anti-inflammatory metabolite is amicrobial amino acid.

In embodiments, the pro-inflammatory metabolite is a microbial lipid, amicrobial carbohydrate or a microbial amino acid as provided herein. Thepro-inflammatory metabolite may be a microbial lipid (e.g.,dihydroxyoctadec-12-enoic acid, cholate or methylmalonate), a microbialcarbohydrate (e.g., n-acetylymuramate, lactobionate or maltotriosesyllactose) or a microbial amino acid (e.g., ornithine or taurine) asprovided herein.

In embodiments, the inflammatory disease is asthma, ulcerative colitis,irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, orerythema nodosum. In embodiments, the inflammatory disease is asthma. Inembodiments, the inflammatory disease is ulcerative colitis. Inembodiments, the inflammatory disease is irritable bowel syndrome. Inembodiments, the inflammatory disease is arthritis. In embodiments, theinflammatory disease is uveitis. In embodiments, the inflammatorydisease is pyoderma gangrenosum. In embodiments, the inflammatorydisease is erythema nodosum.

In an aspect, a method of monitoring the effect of treatment for aninflammatory disease in a subject undergoing inflammatory diseasetherapy or a patient that has received inflammatory disease therapy isprovided. The method includes (i) determining a first expression levelof an anti-inflammatory metabolite in the subject at a first time point;(ii) determining a second expression level of an anti-inflammatorymetabolite in the subject at a second time point; and (iii) comparingthe second expression level of an anti-inflammatory metabolite to thefirst expression level of an anti-inflammatory metabolite, therebydetermining the effect of treatment for an inflammatory disease in thesubject.

In embodiments, the anti-inflammatory metabolite is a microbial lipid, amicrobial carbohydrate or a microbial amino acid as provided herein. Inembodiments, the anti-inflammatory metabolite is a microbial lipid. Inembodiments, the anti-inflammatory metabolite is a microbialcarbohydrate. In embodiments, the anti-inflammatory metabolite is amicrobial amino acid.

In an aspect, a method of monitoring the effect of treatment for aninflammatory disease in a subject undergoing inflammatory diseasetherapy or a patient that has received inflammatory disease therapy isprovided. The method includes (i) determining a first expression levelof a pro-inflammatory metabolite in the subject at a first time point;(ii) determining a second expression level of a pro-inflammatorymetabolite in the subject at a second time point; and (iii) comparingthe second expression level of a pro-inflammatory metabolite to thefirst expression level of a pro-inflammatory metabolite, therebydetermining the effect of treatment for an inflammatory disease in thesubject.

In embodiments, the pro-inflammatory metabolite is a microbial lipid, amicrobial carbohydrate or a microbial amino acid as provided herein. Inembodiments, the pro-inflammatory metabolite is a microbial lipid. Inembodiments, the pro-inflammatory metabolite is a microbialcarbohydrate. In embodiments, the pro-inflammatory metabolite is amicrobial amino acid.

In embodiments, the inflammatory disease is asthma, ulcerative colitis,irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, orerythema nodosum. In embodiments, the inflammatory disease is asthma. Inembodiments, the inflammatory disease is ulcerative colitis. Inembodiments, the inflammatory disease is irritable bowel syndrome. Inembodiments, the inflammatory disease is arthritis. In embodiments, theinflammatory disease is uveitis. In embodiments, the inflammatorydisease is pyoderma gangrenosum. In embodiments, the inflammatorydisease is erythema nodosum.

In an aspect, a method of determining whether a subject has or is atrisk of developing dysbiosis or an inflammatory disease is provided. Themethod including: (i) obtaining a biological sample from the subject;and (ii) detecting whether the biological sample is pro-inflammatory.

In embodiments, the subject suffers from or resides with someone whosuffers from a bacterial, viral, or fungal gastrointestinal infection.

In embodiments, the subject (i) has at least 1, 2, 3, or 4 cousins,grandparents, parents, aunts, uncles, and/or siblings who have beendiagnosed with an inflammatory disease; (ii) suffers from constipation,diarrhea, bloating, urgency, and/or abdominal pain; and/or (iii) hasbeen administered an antibiotic within the last 1, 2, or 4 months.

In embodiments, the inflammatory disease is an allergy, atopy, asthma,an autoimmune disease, an autoinflammatory disease, a hypersensitivity,pediatric allergic asthma, allergic asthma, inflammatory bowel disease,Celiac disease, Crohn's disease, colitis, ulcerative colitis,collagenous colitis, lymphocytic colitis, diverticulitis, irritablebowel syndrome, short bowel syndrome, stagnant loop syndrome, chronicpersistent diarrhea, intractable diarrhea of infancy, Traveler'sdiarrhea, immunoproliferative small intestinal disease, chronicprostatitis, postenteritis syndrome, tropical sprue, Whipple's disease,Wolman disease, arthritis, rheumatoid arthritis, Behçet's disease,uveitis, pyoderma gangrenosum, erythema nodosum, traumatic brain injury,psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis,systemic lupus erythematosus (SLE), myasthenia gravis, juvenile onsetdiabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto'sencephalitis, Hashimoto's thyroiditis, ankylosing spondylitis,psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis,auto-immune thyroiditis, bullous pemphigoid, sarcoidosis, ichthyosis,Graves ophthalmopathy, Addison's disease, Vitiligo, acne vulgaris,pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplantrejection, interstitial cystitis, atherosclerosis, and atopicdermatitis.

In embodiments, the subject is less than about 1, 2, 3, 4, 5, 6, 7, 8,9, 12, 18, or 24 months old. In embodiments, the subject is less thanabout 1 month old. In embodiments, the subject is less than 1 month old.In embodiments, the subject is less than about 2 months old. Inembodiments, the subject is less than 2 months old. In embodiments, thesubject is less than about 3 months old. In embodiments, the subject isless than 3 months old. In embodiments, the subject is less than about 4months old. In embodiments, the subject is less than 4 months old. Inembodiments, the subject is less than about 5 months old. Inembodiments, the subject is less than 5 months old. In embodiments, thesubject is less than about 6 months old. In embodiments, the subject isless than 6 months old. In embodiments, the subject is less than about 7months old. In embodiments, the subject is less than 7 months old. Inembodiments, the subject is less than about 8 months old. Inembodiments, the subject is less than 8 months old. In embodiments, thesubject is less than about 9 months old. In embodiments, the subject isless than 9 months old. In embodiments, the subject is less than about12 months old. In embodiments, the subject is less than 12 months old.In embodiments, the subject is less than about 18 months old. Inembodiments, the subject is less than 18 months old. In embodiments, thesubject is less than about 24 months old. In embodiments, the subject isless than 24 months old.

In embodiments, the subject is between about 2 and about 18 years old,or is at least about 18 years old. In embodiments, the subject isbetween 2 and 18 years old, or is at least 18 years old. In embodiments,the subject is between about 2 and about 18 years old, or is at leastabout 18 (e.g., 19, 20, 25, 30, 40, 50, 60, 70, 80, 90) years old. Inembodiments, the subject is between about 2 and about 18 years old, oris about 19 years old. In embodiments, the subject is between about 2and about 18 years old, or is 19 years old. In embodiments, the subjectis between about 2 and about 18 years old, or is about 20 years old. Inembodiments, the subject is between about 2 and about 18 years old, oris 20 years old. In embodiments, the subject is between about 2 andabout 18 years old, or is about 25 years old. In embodiments, thesubject is between about 2 and about 18 years old, or is 25 years old.In embodiments, the subject is between about 2 and about 18 years old,or is about 30 years old. In embodiments, the subject is between about 2and about 18 years old, or is 30 years old. In embodiments, the subjectis between about 2 and about 18 years old, or is about 40 years old. Inembodiments, the subject is between about 2 and about 18 years old, oris 40 years old. In embodiments, the subject is between about 2 andabout 18 years old, or is about 50 years old. In embodiments, thesubject is between about 2 and about 18 years old, or is 50 years old.In embodiments, the subject is between about 2 and about 18 years old,or is about 60 years old. In embodiments, the subject is between about 2and about 18 years old, or is 60 years old. In embodiments, the subjectis between about 2 and about 18 years old, or is about 70 years old. Inembodiments, the subject is between about 2 and about 18 years old, oris 70 years old. In embodiments, the subject is between about 2 andabout 18 years old, or is about 80 years old. In embodiments, thesubject is between about 2 and about 18 years old, or is 80 years old.In embodiments, the subject is between about 2 and about 18 years old,or is about 90 years old. In embodiments, the subject is between about 2and about 18 years old, or is 90 years old.

In embodiments, the subject comprises a gastrointestinal microbiome that(a) has an increased proportion of Streptococcus spp., Bifidobacteriumspp., and Enterococcus spp. compared to a healthy or general population;(b) has a reduced proportion of Alternaria alternata, Aspergillusflavus, Aspergillus cibarius, and Candida sojae compared to a healthy orgeneral population; (c) has an increased proportion of Candida albicansand Debaryomyces spp. compared to a healthy or general population; (d)has a reduced proportion of Bifidobacteria spp., Lactobacillus spp.,Faecalibacterium spp. and Akkermansia spp. compared to a healthy orgeneral population; (e) has a reduced proportion of Malassezia spp.compared to a healthy or general population; (f) has an increasedproportion of Bacterioides spp., Ruminococcus spp., Prevotella spp., orBifidobacterium spp. compared to a healthy or general population; or (g)has an increased proportion of Enterococcus faecalis, Enterococcusfaecium, or Clostridium difficile compared to a healthy or generalpopulation.

In embodiments, the biological sample is a bodily fluid. In embodiments,the bodily fluid is blood, plasma, serum, fecal water, or abrancheoaleolar lavage. In embodiments, the bodily fluid is fecal water.

In embodiments, detecting whether the biological sample ispro-inflammatory includes contacting an antigen presenting cell with thebiological sample. In embodiments, the antigen presenting cell is adendritic cell.

In embodiments, detecting whether the biological sample ispro-inflammatory further includes contacting a naïve T cell with theantigen presenting cell to produce a contacted T cell.

In embodiments, the method further includes detecting a cytokineproduced by the contacted T cell and/or the progeny of the contacted Tcell.

In embodiments, the biological sample is detected to be pro-inflammatoryif (i) the proportion of T-helper (TH)-2 cells is increased in theprogeny of the contacted T cell compared to a control; (ii) theproportion of TH-1, TH-17, and/or TH22 cells is increased in the progenyof the contacted T cell compared to a control; (iii) the ratio of TH-1cells to TH-2 cells is decreased in the progeny of the contacted T cellcompared to a control; (iv) the proportion of IL-17 producing CD8+ Tcells is increased in the progeny of the contacted T cell compared to acontrol; and/or (v) the amount of IL-4, IL-10, and/or IL-13 produced bythe progeny of the contacted T cell and/or the progeny thereof isincreased compared to a control.

In embodiments, the control is (i) the corresponding proportion, ratio,and/or amount of a corresponding T cell that has been contacted withsterile culture medium and/or the progeny thereof; (ii) thecorresponding proportion, ratio, and/or amount of a corresponding T cellthat has been contacted with an antigen presenting cell that has beencontacted with a biological sample from a subject who does not havedysbiosis, an inflammatory disease, or a gastrointestinal infection,and/or the progeny thereof, and/or (iii) a reference value correspondingto the proportion, ratio, and/or amount in the general population or apopulation of subjects who do not have dysbiosis, an inflammatorydisease, or a gastrointestinal infection.

In embodiments, the method further includes directing the subject toreceive treatment or further testing or monitoring for dysbiosis or aninflammatory disease if the biological sample is detected to bepro-inflammatory.

In embodiments, the method further includes administering a bacterialpopulation or composition as described herein including embodimentsthereof to the subject if the biological sample is detected to bepro-inflammatory.

In embodiments, the subject includes a gastrointestinal microbiome that(a) has an increased proportion of Streptococcus spp., Bifidobacteriumspp., and Enterococcus spp. compared to a healthy or general population;(b) has a reduced proportion of Alternaria alternata, Aspergillusflavus, Aspergillus cibarius, and Candida sojae compared to a healthy orgeneral population; (c) has an increased proportion of Candida albicansand Debaryomyces spp. compared to a healthy or general population; (d)has a reduced proportion of Bifidobacteria spp., Lactobacillus spp.,Faecalibacterium spp. and Akkermansia spp. compared to a healthy orgeneral population; (e) has a reduced proportion of Malassezia spp.compared to a healthy or general population; (f) has an increasedproportion of Bacterioides spp., Ruminococcus spp., Prevotella spp., orBifidobacterium spp. compared to a healthy or general population; or (g)has an increased proportion of Enterococcus faecalis, Enterococcusfaecium, or Clostridium difficile compared to a healthy or generalpopulation.

In embodiments, the method further includes determining whether thesubject has a gastrointestinal microbiome that (a) has an increasedproportion of Streptococcus spp., Bifidobacterium spp., and Enterococcusspp. compared to a healthy or general population; (b) has a reducedproportion of Alternaria alternata, Aspergillus flavus, Aspergilluscibarius, and Candida sojae compared to a healthy or general population;(c) has an increased proportion of Candida albicans and Debaryomycesspp. compared to a healthy or general population; (d) has a reducedproportion of Bifidobacteria spp., Lactobacillus spp., Faecalibacteriumspp. and Akkermansia spp. compared to a healthy or general population;(e) has a reduced proportion of Malassezia spp. compared to a healthy orgeneral population; (f) has an increased proportion of Bacterioidesspp., Ruminococcus spp., Prevotella spp., or Bifidobacterium spp.compared to a healthy or general population; or (g) has an increasedproportion of Enterococcus faecalis, Enterococcus faecium, orClostridium difficile compared to a healthy or general population.

In embodiments, a method of treating or preventing dysbiosis, a viralrespiratory infection, or an inflammatory disease in a subjectdetermined to have or be at risk of developing dysbiosis, a viralrespiratory infection, or an inflammatory disease according to a methoddescribed herein including embodiments thereof. In embodiments, themethod includes administering a bacterial population disclosed herein tothe subject.

In another aspect, a method of determining an inflammatory diseaseactivity in a subject is provided. The method includes (i) detecting anexpression level of one or more anti-inflammatory metabolites in asubject; (ii) determining whether the expression level is modulatedrelative to a standard control, thereby determining an inflammatorydisease activity in the subject; and (iii) based at least in part on theexpression level in step (ii), determining the inflammatory diseaseactivity in the subject.

In embodiments, the anti-inflammatory metabolite is a microbial lipid, amicrobial carbohydrate or a microbial amino acid as provided herein. Inembodiments, the anti-inflammatory metabolite is a microbial lipid. Inembodiments, the anti-inflammatory metabolite is a microbialcarbohydrate. In embodiments, the anti-inflammatory metabolite is amicrobial amino acid.

In another aspect, a method of determining an inflammatory diseaseactivity in a subject is provided. The method includes (i) detecting anexpression level of one or more pro-inflammatory metabolites in asubject; (ii) determining whether the expression level is modulatedrelative to a standard control, thereby determining an inflammatorydisease activity in the subject; and (iii) based at least in part on theexpression level in step (ii), determining the inflammatory diseaseactivity in the subject.

In embodiments, the pro-inflammatory metabolite is a microbial lipid, amicrobial carbohydrate or a microbial amino acid as provided herein. Inembodiments, the anti-inflammatory metabolite is a microbial lipid. Inembodiments, the anti-inflammatory metabolite is a microbialcarbohydrate. In embodiments, the anti-inflammatory metabolite is amicrobial amino acid.

In embodiments, the inflammatory disease is asthma, ulcerative colitis,irritable bowel syndrome, arthritis, uveitis, pyoderma gangrenosum, orerythema nodosum. In embodiments, the inflammatory disease is asthma. Inembodiments, the inflammatory disease is ulcerative colitis. Inembodiments, the inflammatory disease is irritable bowel syndrome. Inembodiments, the inflammatory disease is arthritis. In embodiments, theinflammatory disease is uveitis. In embodiments, the inflammatorydisease is pyoderma gangrenosum. In embodiments, the inflammatorydisease is erythema nodosum.

In an aspect, a method of determining whether a subject has or is atrisk of developing dysbiosis or an inflammatory disease is provided. Themethod including: (i) detecting an expression level of one or moreanti-inflammatory metabolites or pro-inflammatory metabolites in asubject; (ii) determining whether the expression level is increased ordecreased relative to a standard control, wherein an elevated expressionlevel of an pro-inflammatory metabolite or a decreased expression levelof an anti-inflammatory metabolite relative to the standard controlindicates that the subject has or is at risk of developing aninflammatory disease, and (iii) based at least in part on the expressionlevel in step (ii), determining whether the subject has or is at riskfor developing an inflammatory disease.

In an aspect, a method of monitoring the effect of treatment for aninflammatory disease in a subject undergoing inflammatory diseasetherapy or a patient that has received inflammatory disease therapyincluding: (i) determining a first expression level of ananti-inflammatory or pro-inflammatory metabolite in the subject at afirst time point; (ii) determining a second expression level of ananti-inflammatory or pro-inflammatory metabolite in the subject at asecond time point; and (iii) comparing the second expression level of ananti-inflammatory or pro-inflammatory metabolite to the first expressionlevel of an anti-inflammatory or pro-inflammatory metabolite, therebydetermining the effect of treatment for an inflammatory disease in thesubject is provided.

In an aspect, a method of determining an inflammatory disease activityin a subject is provided. The method including: (i) detecting anexpression level of one or more anti-inflammatory or pro-inflammatorymetabolites in a subject; (ii) determining whether the expression levelis modulated relative to a standard control, thereby determining aninflammatory disease activity in the subject; and (iii) based at leastin part on the expression level in step (ii), determining theinflammatory disease activity in the subject.

TABLE 1 Non-limiting examples of Lactobacillus sp., Faecalibacteriumsp., Akkermansia sp., Myxococcus sp., Cystobacter sp., and Pediococcussp. that can be used singly, or in any combination in bacterialpopulations of methods and compositions provided herein. Phylum ClassOrder Family Genus Species Verruco- Verruco- Verruco- Verruco-Akkermansia Akkermansia microbia microbiae microbiales microbiaceaemuciniphila Firmicutes Clostridia Clostridiales Rumino- Faecali-Faecali- coccaceae bacterium bacterium prausnitzii Proteo- Deltaproteo-Myxococcales unclassified sfA unclassified bacteria bacteria Proteo-Deltaproteo- Myxococcales Cystobacteraceae Cystobacter Cystobacterbacteria bacteria fuscus Proteo- Deltaproteo- Myxococcales MyxococcaceaeMyxococcus Myxococcus bacteria bacteria xanthus Firmicutes BacilliLactobacillales Lactobacillaceae Lactobacillus Lactobacillus zeae (Lactobacillus rhamnosus) Firmicutes Bacilli Lactobacillales LactobacillaceaeLactobacillus Lactobacillus acidipiscis Firmicutes BacilliLactobacillales Lactobacillaceae Lactobacillus Lactobacillus acidophilusFirmicutes Bacilli Lactobacillales Lactobacillaceae LactobacillusLactobacillus agilis Firmicutes Bacilli Lactobacillales LactobacillaceaeLactobacillus Lactobacillus aviarius Firmicutes Bacilli LactobacillalesLactobacillaceae Lactobacillus Lactobacillus brevis Firmicutes BacilliLactobacillales Lactobacillaceae Lactobacillus Lactobacilluscoleohominis Firmicutes Bacilli Lactobacillales LactobacillaceaeLactobacillus Lactobacillus crispatus Firmicutes Bacilli LactobacillalesLactobacillaceae Lactobacillus Lactobacillus crustorum FirmicutesBacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacilluscurvatus Firmicutes Bacilli Lactobacillales LactobacillaceaeLactobacillus Lactobacillus diolivorans Firmicutes BacilliLactobacillales Lactobacillaceae Lactobacillus Lactobacillus farraginisFirmicutes Bacilli Lactobacillales Lactobacillaceae LactobacillusLactobacillus fermentum Firmicutes Bacilli LactobacillalesLactobacillaceae Lactobacillus Lactobacillus fuchuensis FirmicutesBacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillusharbinensis Firmicutes Bacilli Lactobacillales LactobacillaceaeLactobacillus Lactobacillus helveticus Firmicutes BacilliLactobacillales Lactobacillaceae Lactobacillus Lactobacillus hilgardiiFirmicutes Bacilli Lactobacillales Lactobacillaceae LactobacillusLactobacillus intestinalis Firmicutes Bacilli LactobacillalesLactobacillaceae Lactobacillus Lactobacillus jensenii Firmicutes BacilliLactobacillales Lactobacillaceae Lactobacillus Lactobacillus johnsoniiFirmicutes Bacilli Lactobacillales Lactobacillaceae LactobacillusLactobacillus kefiranofaciens Firmicutes Bacilli LactobacillalesLactobacillaceae Lactobacillus Lactobacillus kefiri Firmicutes BacilliLactobacillales Lactobacillaceae Lactobacillus Lactobacillus lindneriFirmicutes Bacilli Lactobacillales Lactobacillaceae LactobacillusLactobacillus mali Firmicutes Bacilli Lactobacillales LactobacillaceaeLactobacillus Lactobacillus manihotivorans Firmicutes BacilliLactobacillales Lactobacillaceae Lactobacillus Lactobacillus mucosaeFirmicutes Bacilli Lactobacillales Lactobacillaceae LactobacillusLactobacillus oeni Firmicutes Bacilli Lactobacillales LactobacillaceaeLactobacillus Lactobacillus oligofermentans Firmicutes BacilliLactobacillales Lactobacillaceae Lactobacillus Lactobacillus panisFirmicutes Bacilli Lactobacillales Lactobacillaceae LactobacillusLactobacillus pantheris Firmicutes Bacilli LactobacillalesLactobacillaceae Lactobacillus Lactobacillus parabrevis FirmicutesBacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillusparacollinoides Firmicutes Bacilli Lactobacillales LactobacillaceaeLactobacillus Lactobacillus parakefiri Firmicutes BacilliLactobacillales Lactobacillaceae Lactobacillus Lactobacillusparaplantarum Firmicutes Bacilli Lactobacillales LactobacillaceaeLactobacillus Lactobacillus pentosus Firmicutes Bacilli LactobacillalesLactobacillaceae Lactobacillus Lactobacillus pontis Firmicutes BacilliLactobacillales Lactobacillaceae Lactobacillus Lactobacillus reuteriFirmicutes Bacilli Lactobacillales Lactobacillaceae LactobacillusLactobacillus rossiae Firmicutes Bacilli LactobacillalesLactobacillaceae Lactobacillus Lactobacillus salivarius FirmicutesBacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillussiliginis Firmicutes Bacilli Lactobacillales LactobacillaceaeLactobacillus Lactobacillus sucicola Firmicutes Bacilli LactobacillalesLactobacillaceae Lactobacillus Lactobacillus vaccinostercus FirmicutesBacilli Lactobacillales Lactobacillaceae Lactobacillus Lactobacillusvaginalis Firmicutes Bacilli Lactobacillales LactobacillaceaeLactobacillus Lactobacillus vini Firmicutes Bacilli LactobacillalesLactobacillaceae Lactobacillus Lactococcus garvieae Firmicutes BacilliLactobacillales Lactobacillaceae Lactobacillus Lactococcus lactisFirmicutes Bacilli Lactobacillales Lactobacillaceae PediococcusPediococcus pentosaceus Firmicutes Bacilli LactobacillalesLactobacillaceae Pediococcus Pediococcus acidilactici Firmicutes BacilliLactobacillales Lactobacillaceae Pediococcus Pediococcus damnosusFirmicutes Bacilli Lactobacillales Lactobacillaceae PediococcusPediococcus ethanolidurans Firmicutes Bacilli LactobacillalesLactobacillaceae Pediococcus Pediococcus parvulus ActinobacteriaActinobacteria Bifidobacteriales Bifidobacteriaceae BifidobacteriumBifidobacterium bifidum Actinobacteria Actinobacteria BifidobacterialesBifidobacteriaceae Bifidobacterium Bifidobacterium pseudolongumActinobacteria Actinobacteria Bifidobacteriales BifidobacteriaceaeBifidobacterium Bifidobacterium saeculare Actinobacteria ActinobacteriaBifidobacteriales Bifidobacteriaceae Bifidobacterium Bifidobacteriumsubtile Firmicutes Clostridia Clostridiales Clostridiaceae ClostridiumClostridium hiranonis

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

EMBODIMENTS

Embodiments include P1 to P34 following.

Embodiment P1

A method of treating or preventing an inflammatory disease in a subjectin need thereof, said method comprising administering to said subject atherapeutically effective amount of Lactobacillus johnsonii,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus and Pediococcus pentosaceus.

Embodiment P2

The method of embodiment 1, further comprising a pharmaceutically activeexcipient.

Embodiment P3

The method of embodiment 1 or 2, wherein said Lactobacillus johnsonii.Faecalibacterium prausnitzii, Akkermansia muciniphila. Myxococcusxanthus and Pediococcus pentosaceus form a microbial composition.

Embodiment P4

The method of embodiment 3, wherein said microbial composition iseffective for administration to the gut.

Embodiment P5

The method of embodiment 3, wherein said microbial composition iseffective to increase an anti-inflammatory metabolite.

Embodiment P6

The method of embodiment 3, wherein said microbial composition iseffective to decrease a pro-inflammatory metabolite.

Embodiment P7

The method of embodiment 5, wherein said anti-inflammatory metabolite isa microbial lipid, a microbial carbohydrate or a microbial amino acid.

Embodiment P8

The method of embodiment 6, wherein said pro-inflammatory metabolite isa microbial lipid, a microbial carbohydrate or a microbial amino acid.

Embodiment P9

The method of embodiment 8, wherein said pro-inflammatory metabolite isIL-4. IL-10, IL-13 or MUC5B.

Embodiment P10

The method of one of embodiments 1 or 9, wherein said Lactobacillusjohnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila,Myxococcus xanthus and Pediococcus pentosaceus are metabolically active.

Embodiment P11

The method of one of embodiment 1 or 9, wherein said Lactobacillusjohnsonii, Faecalibacterium prausnitzii, Akkermansia muciniphila,Myxococcus xanthus and Pediococcus pentosaceus are metabolicallyinactive.

Embodiment P12

The method of one of embodiments 1-11, further comprising administeringa therapeutically effective amount of a fungus.

Embodiment P13

The method of one of embodiments 1-12, wherein said subject is aneonate.

Embodiment P14

The method of one of embodiments 1-13, wherein said inflammatory diseaseis asthma, ulcerative colitis, irritable bowel syndrome, arthritis,uveitis, pyoderma gangrenosum, or erythema nodosum.

Embodiment P15

A method of increasing an anti-inflammatory metabolite in a subject inneed thereof, the method comprising administering to said subject atherapeutically effective amount of Lactobacillus johnsonii,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus and Pediococcus pentosaceus.

Embodiment P16

The method of embodiment 15, further comprising a pharmaceuticallyactive excipient.

Embodiment P17

The method of embodiment 15 or 16, wherein said Lactobacillus johnsonii,Faecalibacterium prausnitzii, Akkermansia muciniphila, Myxococcusxanthus and Pediococcus pentosaceus form a microbial composition.

Embodiment P18

The method of one of embodiments 15-17, wherein said anti-inflammatorymetabolite is a microbial lipid, a microbial carbohydrate or a microbialamino acid.

Embodiment P19

The method of one of embodiments 15-18, wherein said inflammatorydisease is asthma, ulcerative colitis, irritable bowel syndrome,arthritis, uveitis, pyoderma gangrenosum, or erythema nodosum.

Embodiment P20

A method of detecting an anti-inflammatory metabolite in a subject thathas or is at risk for developing an inflammatory disease, said methodcomprising: (i) obtaining a biological sample from said subject; and(ii) determining an expression level of an anti-inflammatory metabolitein said biological sample.

Embodiment P21

The method of embodiment 20, wherein said anti-inflammatory metaboliteis a microbial lipid or a microbial carbohydrate.

Embodiment P22

The method of embodiment 20 or 21, wherein said biological sample is abodily fluid.

Embodiment P23

The method of embodiment 22, wherein said bodily fluid is serum, fecalwater or brancheoaleolar lavage.

Embodiment P24

The method of one of embodiments 20-23, wherein said determiningcomprises: (a) contacting in vitro said anti-inflammatory metabolitewith an antigen presenting cell, thereby forming a metabolite-antigenpresenting cell; (b) contacting said metabolite-antigen presenting cellwith a T cell, thereby forming a contacted T cell; and (c) detecting acytokine produced by said contacted T cell.

Embodiment P25

The method of one of embodiments 20-24, wherein said inflammatorydisease is asthma, ulcerative colitis, irritable bowel syndrome,arthritis, uveitis, pyoderma gangrenosum, or erythema nodosum.

Embodiment P26

A method of determining whether a subject has or is at risk ofdeveloping an inflammatory disease, said method comprising: (i)detecting an expression level of one or more anti-inflammatorymetabolites or pro-inflammatory metabolites in a subject; (ii)determining whether said expression level is increased or decreasedrelative to a standard control, wherein an elevated expression level ofan pro-inflammatory metabolite or a decreased expression level of ananti-inflammatory metabolite relative to said standard control indicatesthat said subject has or is at risk of developing an inflammatorydisease; and (iii) based at least in part on said expression level instep (ii), determining whether said subject has or is at risk fordeveloping an inflammatory disease.

Embodiment P27

The method of embodiment 26, wherein said inflammatory disease isasthma, ulcerative colitis, irritable bowel syndrome, arthritis,uveitis, pyoderma gangrenosum, or erythema nodosum.

Embodiment P28

The method of embodiment 26, wherein said anti-inflammatory metaboliteis a microbial lipid, a microbial carbohydrate or a microbial aminoacid.

Embodiment P29

A method of monitoring the effect of treatment for an inflammatorydisease in a subject undergoing inflammatory disease therapy or apatient that has received inflammatory disease therapy comprising: (i)determining a first expression level of an anti-inflammatory metabolitein the subject at a first time point; (ii) determining a secondexpression level of an anti-inflammatory metabolite in the subject at asecond time point; and (iii) comparing the second expression level of ananti-inflammatory metabolite to the first expression level of ananti-inflammatory metabolite, thereby determining the effect oftreatment for an inflammatory disease in the subject.

Embodiment P30

The method of embodiment 29, wherein said inflammatory disease isasthma, ulcerative colitis, irritable bowel syndrome, arthritis,uveitis, pyoderma gangrenosum, or erythema nodosum.

Embodiment P31

The method of embodiment 29, wherein said anti-inflammatory metaboliteis a microbial lipid, a microbial carbohydrate or a microbial aminoacid.

Embodiment P32

A method of determining an inflammatory disease activity in a subject,said method comprising: (i) detecting an expression level of one or moreanti-inflammatory metabolites in a subject; (ii) determining whethersaid expression level is modulated relative to a standard control,thereby determining an inflammatory disease activity in said subject;and (iii) based at least in part on said expression level in step (ii),determining said inflammatory disease activity in said subject.

Embodiment P33

The method of embodiment 32, wherein said inflammatory disease isasthma, ulcerative colitis, irritable bowel syndrome, arthritis,uveitis, pyoderma gangrenosum, or erythema nodosum.

Embodiment P34

The method of embodiment 32, wherein said anti-inflammatory metaboliteis a microbial lipid, a microbial carbohydrate or a microbial aminoacid.

Further embodiments include embodiments 1 to 111 following.

Embodiment 1

A method of treating or preventing dysbiosis in a subject in needthereof, the method comprising administering to the subject an effectiveamount of a bacterial population comprising Lactobacillus sp.,Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and Pediococcussp.

Embodiment 2

The method of embodiment 1, wherein (i) the Lactobacillus sp. isLactobacillus johnsonii; (ii) the Faecalibacterium sp., isFaecalibacterium prausnitzii; (iii) the Akkermansia sp. is Akkermansiamuciniphila; (iv) the Myxococcus sp. is Myxococcus xanthus; and (v) thePediococcus sp. is Pediococcus pentosaceus.

Embodiment 3

The method of embodiment 1 or 2, wherein (i) the Lactobacillus sp. isLactobacillus zeae, Lactobacillus acidipiscis, Lactobacillusacidophilus, Lactobacillus agilis, Lactobacillus aviarius, Lactobacillusbrevis, Lactobacillus coleohominis, Lactobacillus crispatus,Lactobacillus crustorum, Lactobacillus curvatus, Lactobacillusdiolivorans, Lactobacillus farraginis, Lactobacillus fermentum,Lactobacillus fuchuensis, Lactobacillus harbinensis, Lactobacillushelveticus, Lactobacillus hilgardii, Lactobacillus intestinalis,Lactobacillus jensenii, Lactobacillus kefiranofaciens, Lactobacilluskefiri, Lactobacillus lindneri, Lactobacillus mall, Lactobacillusmanihotivorans, Lactobacillus mucosae, Lactobacillus oeni, Lactobacillusoligofermentans, Lactobacillus panis, Lactobacillus pantheris,Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillusparakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus,Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae,Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillussucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis; (ii)the Faecalibacterium sp., is Faecalibacterium prausnitzii; (iii) theAkkermansia sp. is Akkermansia muciniphila; (iv) the Myxococcus sp. isMyxococcus xanthus; and (v) the Pediococcus sp. is Pediococcuspentosaceus, Pediococcus acidilactici, Pediococcus damnosus, Pediococcusethanolidurans, or Pediococcus parvulus.

Embodiment 4

The method of any one of embodiments 1 to 3, wherein the Myxococcus sp.is in the form of spores, vegetative bacteria, or a mixture of sporesand vegetative bacteria.

Embodiment 5

The method of embodiment 4, wherein the Myxococcus sp. is in the form ofa powder comprising spores.

Embodiment 6

The method of any one of embodiments 1 to 5, wherein less than about 20,15, 10, 9, 8, 7, or 6 different species of bacteria are administered tothe subject.

Embodiment 7

The method of embodiment 1, wherein the bacterial population forms partof a bacterial composition.

Embodiment 8

The method of embodiment 7, wherein the bacterial composition comprisesless than about 20, 15, 10, 9, 8, 7, or 6 species of bacteria.

Embodiment 9

The method of embodiment 7 or 8, wherein the bacterial composition isnot a fecal transplant.

Embodiment 10

The method of any one of embodiments 7 to 9, wherein the bacterialcomposition further comprises a pharmaceutically acceptable excipient.

Embodiment 11

The method of any one of embodiments 7 to 10, wherein the bacterialcomposition is a capsule, a tablet, a suspension, a suppository, apowder, a cream, an oil, an oil-in-water emulsion, a water-in-oilemulsion, or an aqueous solution.

Embodiment 12

The method of any one of embodiments 7 to 10, wherein the bacterialcomposition is in the form of a powder, a solid, a semi-solid, or aliquid.

Embodiment 13

The method of any one of embodiments 7 to 12, wherein the bacterialcomposition has a water activity (a_(w)) less than about 0.9, 0.8, 0.7,0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 at 20° C.

Embodiment 14

The method of any one of embodiments 7 to 13, wherein the bacterialcomposition is a food or a beverage.

Embodiment 15

The method of any one of embodiments 7 to 14, wherein the bacterialcomposition is administered orally or rectally.

Embodiment 16

The method of any one of embodiments 1 to 15, wherein the Lactobacillussp., the Faecalibacterium sp., the Akkermansia sp., the Myxococcus sp.,and/or the Pediococcus sp. is in the form of a powder.

Embodiment 17

The method of any one of embodiments 1 to 16, wherein the Lactobacillussp., the Faecalibacterium sp., the Akkermansia sp., the Myxococcus sp.,and/or the Pediococcus sp. has been lyophilized.

Embodiment 18

The method of any one of embodiments 1 to 17, wherein the subject is ahuman.

Embodiment 19

The method of any one of embodiments 1 to 18, wherein the subjectsuffers from or resides with someone who suffers from a bacterial,viral, or fungal gastrointestinal infection.

Embodiment 20

The method of any one of embodiments 1 to 19, wherein the subject has aninflammatory disease.

Embodiment 21

The method of any one of embodiments 1 to 20, wherein the subject is atrisk of suffering from an inflammatory disease.

Embodiment 22

The method of any one of embodiments 1 to 21, wherein the subject has atleast 1, 2, 3, or 4 cousins, grandparents, parents, aunts, uncles,and/or siblings who have been diagnosed with an inflammatory disease.

Embodiment 23

The method of any one of embodiments 20 to 22, wherein the inflammatorydisease is an allergy, atopy, asthma, an autoimmune disease, anautoinflammatory disease, a hypersensitivity, pediatric allergic asthma,allergic asthma, inflammatory bowel disease, Celiac disease, Crohn'sdisease, colitis, ulcerative colitis, collagenous colitis, lymphocyticcolitis, diverticulitis, irritable bowel syndrome, short bowel syndrome,stagnant loop syndrome, chronic persistent diarrhea, intractablediarrhea of infancy, Traveler's diarrhea, immunoproliferative smallintestinal disease, chronic prostatitis, postenteritis syndrome,tropical sprue, Whipple's disease, Wolman disease, arthritis, rheumatoidarthritis, Behçet's disease, uveitis, pyoderma gangrenosum, erythemanodosum, traumatic brain injury, psoriatic arthritis, juvenileidiopathic arthritis, multiple sclerosis, systemic lupus erythematosus(SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitustype 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto'sthyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome,vasculitis, glomerulonephritis, auto-immune thyroiditis, bullouspemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison'sdisease, Vitiligo, acne vulgaris, pelvic inflammatory disease,reperfusion injury, sarcoidosis, transplant rejection, interstitialcystitis, atherosclerosis, and atopic dermatitis.

Embodiment 24

The method of embodiment 23, wherein the inflammatory disease ispediatric allergic asthma or inflammatory bowel disease.

Embodiment 25

The method of any one of embodiments 1 to 24, wherein the subjectsuffers from constipation, diarrhea, bloating, urgency, and/or abdominalpain.

Embodiment 26

The method of any one of embodiments 1 to 25, wherein the subject hasbeen administered an antibiotic within the last 1, 2, 3, or 4 months.

Embodiment 27

The method of any one of embodiments 1 to 26, wherein the subject is aneonate.

Embodiment 28

The method of any one of embodiments 1 to 26, wherein the subject isless than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 18, or 24 months old.

Embodiment 29

The method of any one of embodiments 1 to 26, wherein the subject isbetween about 2 and about 18 years old, or is at least about 18 yearsold.

Embodiment 30

The method of any one of embodiments 1 to 29, wherein the subjectcomprises a gastrointestinal microbiome that

-   -   (a) has an increased proportion of Streptococcus spp.,        Bifidobacterium spp., and Enterococcus spp. compared to a        healthy or general population;    -   (b) has a reduced proportion of Alternaria alternata,        Aspergillus flavus, Aspergillus cibarius, and Candida sojae        compared to a healthy or general population;    -   (c) has an increased proportion of Candida albicans and        Debaryomyces spp. compared to a healthy or general population;    -   (d) has a reduced proportion of Bifidobacteria spp.,        Lactobacillus spp., Faecalibacterium spp. and Akkermansia spp.        compared to a healthy or general population;    -   (e) has a reduced proportion of Malassezia spp. compared to a        healthy or general population;    -   (f) has an increased proportion of Bacterioides spp.,        Ruminococcus spp., Prevotella spp., or Bifidobacterium spp.        compared to a healthy or general population; or    -   (g) has an increased proportion of Enterococcus faecalis,        Enterococcus faecium, or Clostridium difficile compared to a        healthy or general population.

Embodiment 31

The method of any one of embodiments 1 to 30, wherein the effectiveamount is effective to

-   -   (i) increase the level of a Bifidobacterium sp., Clostridia sp.        belonging to Clade IV or XIV, a Lachnospira sp., and/or a        Ruminococcus sp. in the subject;    -   (ii) lower the pH in the feces of the subject;    -   (iii) increase the level of lactic acid in the feces of the        subject;    -   (iv) increase the level of circulating itaconate in the subject;    -   (v) treat, reduce, or prevent allergic inflammation in a        subject;    -   (vi) reduce an adaptive immune response in an airway of the        subject;    -   (vii) reduce dendritic cell activation in a        gastrointestinal-associated mesenteric lymoph node;    -   (viii) increase the level of repair macrophages in the lungs,        blood, serum, or plasma of the subject;    -   (ix) increase the level of an anti-inflammatory compound in the        subject;    -   (x) decrease the level of a pro-inflammatory compound in the        subject;    -   (xi) decrease the level of eotaxin expression and/or secretion        in the subject; and/or decrease the level of mucin expression        and/or secretion in the subject.

Embodiment 32

The method of embodiment 31, wherein the effective amount is effectiveto decrease the level of mucin secretion and/or secretion in the lungsof the subject.

Embodiment 33

The method of embodiment 31 or 32, wherein the anti-inflammatorycompound is a cytokine, a microbial lipid, a microbial carbohydrate, ora microbial amino acid.

Embodiment 34

The method of embodiment 33, wherein the anti-inflammatory compound isIL-17.

Embodiment 35

The method of any one of embodiments 31 to 34, wherein thepro-inflammatory compound is a cytokine, a microbial lipid, a microbialcarbohydrate, or a microbial amino acid.

Embodiment 36

The method of embodiment 35, wherein the pro-inflammatory compound isIL-4, IL-10, IL-8, IL-13, TNF-α, or MUC5B.

Embodiment 37

The method of one of embodiments 1 or 36, wherein the Lactobacillus sp.,Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/orPediococcus sp. is metabolically active.

Embodiment 38

The method of one of embodiments 1 or 36, wherein the Lactobacillus sp.,Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and/orPediococcus sp. is metabolically inactive.

Embodiment 39

The method of any one of embodiments 1 to 38, further comprisingadministering (a) a Bifidobacterium sp., (b) Cystobacter sp., or (c) afungal microorganism to the subject.

Embodiment 40

A method of treating or preventing an inflammatory disease in a subjectin need thereof, the method comprising administering to the subject aneffective amount of a bacterial population comprising Lactobacillus sp.,Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and Pediococcussp.

Embodiment 41

The method of embodiment 40, wherein the inflammatory disease is anallergy, atopy, asthma, an autoimmune disease, an autoinflammatorydisease, a hypersensitivity, pediatric allergic asthma, allergic asthma,inflammatory bowel disease, Celiac disease, Crohn's disease, colitis,ulcerative colitis, collagenous colitis, lymphocytic colitis,diverticulitis, irritable bowel syndrome, short bowel syndrome, stagnantloop syndrome, chronic persistent diarrhea, intractable diarrhea ofinfancy, Traveler's diarrhea, immunoproliferative small intestinaldisease, chronic prostatitis, postenteritis syndrome, tropical sprue,Whipple's disease, Wolman disease, arthritis, rheumatoid arthritis,Behçet's disease, uveitis, pyoderma gangrenosum, erythema nodosum,traumatic brain injury, psoriatic arthritis, juvenile idiopathicarthritis, multiple sclerosis, systemic lupus erythematosus (SLE),myasthenia gravis, juvenile onset diabetes, diabetes mellitus type 1,Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto'sthyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome,vasculitis, glomerulonephritis, auto-immune thyroiditis, bullouspemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison'sdisease, Vitiligo, acne vulgaris, pelvic inflammatory disease,reperfusion injury, sarcoidosis, transplant rejection, interstitialcystitis, atherosclerosis, and atopic dermatitis.

Embodiment 42

A method of treating or preventing a viral respiratory infection in asubject in need thereof, the method comprising administering to thesubject an effective amount of a bacterial population comprisingLactobacillus sp., Faecalibacterium sp., Akkermansia sp., Myxococcussp., and Pediococcus sp.

Embodiment 43

The method of embodiment 42, wherein the viral respiratory infection iscaused by a respiratory syncytial virus, an influenza virus, aparainfluenza virus, an adenovirus, a coronavirus, or a rhinovirus.

Embodiment 44

The method of embodiment 42 or 43, wherein the viral respiratoryinfection is bronchiolitis, a cold, croup, or pneumonia.

Embodiment 45

A method of treating or preventing an allergy in a subject in needthereof, the method comprising administering to the subject an effectiveamount of a bacteria population comprising Lactobacillus sp.,Faecalibacterium sp., Akkermansia sp., Myxococcus sp., and Pediococcussp.

Embodiment 46

The method of embodiment 45, wherein the allergy is an allergy to milk,eggs, fish, shellfish, a tree nut, peanuts, wheat, dander from a cat,dog, or rodent, an insect sting, pollen, latex, dust mites, or soybeans.

Embodiment 47

The method of embodiment 45 or 46, wherein the allergy is pediatricallergic asthma, hay fever, or allergic airway sensitization.

Embodiment 48

A method of increasing the level of an anti-inflammatory compound and/ordecreasing the level of a pro-inflammatory compound in a subject in needthereof, comprising administering to the subject an effective amount ofa bacterial population comprising Lactobacillus sp., Faecalibacteriumsp., Akkermansia sp., Myxococcus sp., and Pediococcus sp.

Embodiment 49

The method of embodiment 48, for increasing the level of theanti-inflammatory compound increases and/or decreases the level of thepro-inflammatory compound in the feces, blood, plasma, serum,broncheoalveolar lavage fluid, sweat, saliva, sputum, lymph, spinalfluid, urine, tears, bile, aqueous humour, vitreous humour, aminioticfluid, breast milk, cerebrospinal fluid, cerumen, nasal mucus, phlegm,or sebum of the subject.

Embodiment 50

The method of one of embodiments 48 or 49, wherein the anti-inflammatorycompound is a microbial lipid, a microbial carbohydrate, or a microbialamino acid.

Embodiment 51

The method of any one of embodiments 48 to 50, wherein subject suffersfrom dysbiosis or an inflammatory disease.

Embodiment 52

A composition comprising Lactobacillus sp., Faecalibacterium sp.,Akkermansia sp., Myxococcus sp., and Pediococcus sp.

Embodiment 53

The composition of embodiment 52, wherein (i) the Lactobacillus sp. isLactobacillus johnsonii, (ii) the Faecalibacterium sp., isFaecalibacterium prausnitzii; (iii) the Akkermansia sp. is Akkermansiamuciniphila; (iv) the Myxococcus sp. is Myxococcus xanthus; and (v) thePediococcus sp. is Pediococcus pentosaceus.

Embodiment 54

The composition of embodiment 52 or 53, wherein the compositioncomprises less than about 20, 15, 10, 9, 8, 7, or 6 different species ofbacteria.

Embodiment 55

The composition of any one of embodiments 52 to 54, wherein thecomposition is not a fecal transplant.

Embodiment 56

The composition of any one of embodiments 52 to 55, further comprising apharmaceutically acceptable excipient.

Embodiment 57

The composition of any one of embodiments 52 to 56, which is a capsule,a tablet, a suspension, a suppository, a powder, a cream, an oil, anoil-in-water emulsion, a water-in-oil emulsion, or an aqueous solution.

Embodiment 58

The composition of any one of embodiments 52 to 57, which is in the formof a powder, a solid, a semi-solid, or a liquid.

Embodiment 59

The composition of any one of embodiments 52 to 58, which has a wateractivity (a_(w)) less than about 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2,or 0.1 at 20° C.

Embodiment 60

The composition of any one of embodiments 52 to 59, which is a food or abeverage.

Embodiment 61

The composition of any one of embodiments 52 to 60, wherein theLactobacillus sp., the Faecalibacterium sp., the Akkermansia sp., theMyxococcus sp., and/or the Pediococcus sp. is in the form of a powder.

Embodiment 62

The composition of any one of embodiments 52 to 61, wherein theLactobacillus sp., the Faecalibacterium sp., the Akkermansia sp., theMyxococcus sp., and/or the Pediococcus sp. has been lyophilized.

Embodiment 63

A method of detecting a pro-inflammatory compound in a subject in needthereof, comprising: (i) obtaining a biological sample from the subject;and (ii) detecting the pro-inflammatory compound in the biologicalsample.

Embodiment 64

The method of embodiment 63, wherein the subject has or is at risk fordeveloping dysbiosis.

Embodiment 65

The method of embodiments 63 or 64, wherein the subject has aninflammatory disease.

Embodiment 66

The method of any one of embodiments 63 to 65, wherein the subject is atrisk of suffering from an inflammatory disease.

Embodiment 67

The method of any one of embodiments 63 to 66, wherein the subject

-   -   (i) has at least 1, 2, 3, or 4 cousins, grandparents, parents,        aunts, uncles, and/or siblings who have been diagnosed with an        inflammatory disease;    -   (ii) suffers from constipation, diarrhea, bloating, urgency,        and/or abdominal pain; and/or    -   (iii) has been administered an antibiotic within the last 1, 2,        or 4 months.

Embodiment 68

The method any one of embodiments 63 to 67, wherein the inflammatorydisease is an allergy, atopy, asthma, an autoimmune disease, anautoinflammatory disease, a hypersensitivity, pediatric allergic asthma,allergic asthma, inflammatory bowel disease, Celiac disease, Crohn'sdisease, colitis, ulcerative colitis, collagenous colitis, lymphocyticcolitis, diverticulitis, irritable bowel syndrome, short bowel syndrome,stagnant loop syndrome, chronic persistent diarrhea, intractablediarrhea of infancy, Traveler's diarrhea, immunoproliferative smallintestinal disease, chronic prostatitis, postenteritis syndrome,tropical sprue, Whipple's disease, Wolman disease, arthritis, rheumatoidarthritis, Behçet's disease, uveitis, pyoderma gangrenosum, erythemanodosum, traumatic brain injury, psoriatic arthritis, juvenileidiopathic arthritis, multiple sclerosis, systemic lupus erythematosus(SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitustype 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto'sthyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome,vasculitis, glomerulonephritis, auto-immune thyroiditis, bullouspemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison'sdisease, Vitiligo, acne vulgaris, pelvic inflammatory disease,reperfusion injury, sarcoidosis, transplant rejection, interstitialcystitis, atherosclerosis, and atopic dermatitis.

Embodiment 69

The method of any one of embodiments 63 to 69, wherein the subject isless than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 18, or 24 months old.

Embodiment 70

The method of any one of embodiments 63 to 69, wherein the subject isbetween about 2 and about 18 years old, or is at least about 18 yearsold.

Embodiment 71

The method of any one of embodiments 63 to 70, wherein the subjectcomprises a gastrointestinal microbiome that

-   -   (a) has an increased proportion of Streptococcus spp.,        Bifidobacterium spp., and Enterococcus spp. compared to a        healthy or general population;    -   (b) has a reduced proportion of Alternaria alternata,        Aspergillus flavus, Aspergillus cibarius, and Candida sojae        compared to a healthy or general population;    -   (c) has an increased proportion of Candida albicans and        Debaryomyces spp. compared to a healthy or general population;    -   (d) has a reduced proportion of Bifidobacteria spp.,        Lactobacillus spp., Faecalibacterium spp. and Akkermansia spp.        compared to a healthy or general population;    -   (e) has a reduced proportion of Malassezia spp. compared to a        healthy or general population    -   (f) has an increased proportion of Bacterioides spp.,        Ruminococcus spp., Prevotella spp., or Bifidobacterium spp.        compared to a healthy or general population; or    -   (g) has an increased proportion of Enterococcus faecalis,        Enterococcus faecium, or Clostridium difficile compared to a        healthy or general population.

Embodiment 72

The method of any one of embodiments 63 to 71, wherein the biologicalsample is a bodily fluid.

Embodiment 73

The method of embodiment 72, wherein the bodily fluid is blood, plasma,serum, fecal water, or a brancheoaleolar lavage.

Embodiment 74

The method of embodiment 72 or 73, wherein the bodily fluid is fecalwater.

Embodiment 75

The method of any one of embodiments 63 to 74, wherein detecting thepro-inflammatory compound comprises contacting an antigen presentingcell with the biological sample.

Embodiment 76

The method of embodiment 75, wherein the antigen presenting cell is adendritic cell.

Embodiment 77

The method of any one of embodiments 63 to 74, wherein detecting thepro-inflammatory compound further comprises contacting a naïve T cellwith the antigen presenting cell to produce a contacted T cell.

Embodiment 78

The method of embodiment 77, further comprising detecting a cytokineproduced by the contacted T cell and/or the progeny of the contacted Tcell.

Embodiment 79

The method of any one of embodiments 77 or 78, wherein thepro-inflammatory compound is detected if

-   -   (i) the proportion of T-helper (TH)-2 cells is increased in the        progeny of the contacted T cell compared to a control;    -   (ii) the proportion of TH-1, TH-17, and/or TH22 cells is        increased in the progeny of the contacted T cell compared to a        control;    -   (iii) the ratio of TH-1 cells to TH-2 cells is decreased in the        progeny of the contacted T cell compared to a control;    -   (iv) the proportion of IL-17 producing CD8+ T cells is increased        in the progeny of the contacted T cell compared to a control;        and/or    -   (v) the amount of IL-4, IL-10, and/or IL-13 produced by the        progeny of the contacted T cell and/or the progeny thereof is        increased compared to a control.

Embodiment 80

The method of embodiment 79, wherein the control is (i) thecorresponding proportion, ratio, and/or amount of a corresponding T cellthat has been contacted with sterile culture medium and/or the progenythereof; (ii) the corresponding proportion, ratio, and/or amount of acorresponding T cell that has been contacted with an antigen presentingcell that has been contacted with a biological sample from a subject whodoes not have dysbiosis, an inflammatory disease, or a gastrointestinalinfection, and/or the progeny thereof; and/or (iii) a reference valuecorresponding to the proportion, ratio, and/or amount in the generalpopulation or a population of subjects who do not have dysbiosis, aninflammatory disease, or a gastrointestinal infection.

Embodiment 81

The method of any one of embodiments 63 to 80, further comprisingdirecting the subject to receive treatment or further testing ormonitoring for dysbiosis or an inflammatory disease if thepro-inflammatory compound is detected in the subject.

Embodiment 82

The method of any one of embodiments 63 to 81, further comprisingadministering the composition of any one of embodiments 52 to 62 to thesubject if the pro-inflammatory compound is detected in the subject.

Embodiment 83

The method of any one of embodiments 63 to 82, further comprisingdiagnosing the subject as having or at risk of developing dysbiosis oran inflammatory disease if the pro-inflammatory compound is detected inthe subject.

Embodiment 84

A method of determining whether a subject has or is at risk ofdeveloping dysbiosis or an inflammatory disease, the method comprising:(i) obtaining a biological sample from the subject; and (ii) detecting apro-inflammatory compound according to the method of any one ofembodiments 63 to 80.

Embodiment 85

A method of determining whether a subject has or is at risk ofdeveloping dysbiosis or an inflammatory disease, the method comprising:(i) obtaining a biological sample from the subject; and (ii) detectingwhether the biological sample is pro-inflammatory.

Embodiment 86

The method of embodiment 85, wherein the subject suffers from or resideswith someone who suffers from a bacterial, viral, or fungalgastrointestinal infection.

Embodiment 87

The method of embodiment 85 or 86, wherein the subject

-   -   (i) has at least 1, 2, 3, or 4 cousins, grandparents, parents,        aunts, uncles, and/or siblings who have been diagnosed with an        inflammatory disease;    -   (ii) suffers from constipation, diarrhea, bloating, urgency,        and/or abdominal pain; and/or    -   (iii) has been administered an antibiotic within the last 1, 2,        or 4 months.

Embodiment 88

The method any one of embodiments 85 to 87, wherein the inflammatorydisease is an allergy, atopy, asthma, an autoimmune disease, anautoinflammatory disease, a hypersensitivity, pediatric allergic asthma,allergic asthma, inflammatory bowel disease, Celiac disease, Crohn'sdisease, colitis, ulcerative colitis, collagenous colitis, lymphocyticcolitis, diverticulitis, irritable bowel syndrome, short bowel syndrome,stagnant loop syndrome, chronic persistent diarrhea, intractablediarrhea of infancy, Traveler's diarrhea, immunoproliferative smallintestinal disease, chronic prostatitis, postenteritis syndrome,tropical sprue, Whipple's disease, Wolman disease, arthritis, rheumatoidarthritis, Behçet's disease, uveitis, pyoderma gangrenosum, erythemanodosum, traumatic brain injury, psoriatic arthritis, juvenileidiopathic arthritis, multiple sclerosis, systemic lupus erythematosus(SLE), myasthenia gravis, juvenile onset diabetes, diabetes mellitustype 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto'sthyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome,vasculitis, glomerulonephritis, auto-immune thyroiditis, bullouspemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, Addison'sdisease, Vitiligo, acne vulgaris, pelvic inflammatory disease,reperfusion injury, sarcoidosis, transplant rejection, interstitialcystitis, atherosclerosis, and atopic dermatitis.

Embodiment 89

The method of any one of embodiments 85 to 88, wherein the subject isless than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 18, or 24 months old.

Embodiment 90

The method of any one of embodiments 85 to 88, wherein the subject isbetween about 2 and about 18 years old, or is at least about 18 yearsold.

Embodiment 91

The method of any one of embodiments 85 to 90, wherein the subjectcomprises a gastrointestinal microbiome that

-   -   (a) has an increased proportion of Streptococcus spp.,        Bifidobacterium spp., and Enterococcus spp. compared to a        healthy or general population;    -   (b) has a reduced proportion of Alternaria alternata,        Aspergillus, flavus, Aspergillus cibarius, and Candida sojae        compared to a healthy or general population;    -   (c) has an increased proportion of Candida albicans and        Debaryomyces spp. compared to a healthy or general population;    -   (d) has a reduced proportion of Bifidobacteria spp.,        Lactobacillus spp., Faecalibacterium spp. and Akkermansia spp.        compared to a healthy or general population;    -   (e) has a reduced proportion of Malassezia spp. compared to a        healthy or general population    -   (f) has an increased proportion of Bacterioides spp.,        Ruminococcus spp., Prevotella spp., or Bifidobacterium spp.        compared to a healthy or general population; or    -   (g) has an increased proportion of Enterococcus faecalis,        Enterococcus faecium, or Clostridium difficile compared to a        healthy or general population.

Embodiment 92

The method of any one of embodiments 85 to 91, wherein the biologicalsample is a bodily fluid.

Embodiment 93

The method of embodiment 92, wherein the bodily fluid is blood, plasma,serum, fecal water, or a brancheoaleolar lavage.

Embodiment 94

The method of embodiment 92 or 93, wherein the bodily fluid is fecalwater.

Embodiment 95

The method of any one of embodiments 93 to 94, wherein detecting whetherthe biological sample is pro-inflammatory comprises contacting anantigen presenting cell with the biological sample.

Embodiment 96

The method of embodiment 95, wherein the antigen presenting cell is adendritic cell.

Embodiment 97

The method of embodiment 95 or 96, wherein detecting whether thebiological sample is pro-inflammatory further comprises contacting anaïve T cell with the antigen presenting cell to produce a contacted Tcell.

Embodiment 98

The method of embodiment 97, further comprising detecting a cytokineproduced by the contacted T cell and/or the progeny of the contacted Tcell.

Embodiment 99

The method of embodiments 97 or 98, wherein biological sample isdetected to be pro-inflammatory if

-   -   (i) the proportion of T-helper (TH)-2 cells is increased in the        progeny of the contacted T cell compared to a control;    -   (ii) the proportion of TH-1, TH-17, and/or TH22 cells is        increased in the progeny of the contacted T cell compared to a        control;    -   (iii) the ratio of TH-1 cells to TH-2 cells is decreased in the        progeny of the contacted T cell compared to a control;    -   (iv) the proportion of IL-17 producing CD8+ T cells is increased        in the progeny of the contacted T cell compared to a control;        and/or    -   (v) the amount of IL-4, IL-10, and/or IL-13 produced by the        progeny of the contacted T cell and/or the progeny thereof is        increased compared to a control.

Embodiment 100

The method of embodiment 99, wherein the control is (i) thecorresponding proportion, ratio, and/or amount of a corresponding T cellthat has been contacted with sterile culture medium and/or the progenythereof; (ii) the corresponding proportion, ratio, and/or amount of acorresponding T cell that has been contacted with an antigen presentingcell that has been contacted with a biological sample from a subject whodoes not have dysbiosis, an inflammatory disease, or a gastrointestinalinfection, and/or the progeny thereof; and/or (iii) a reference valuecorresponding to the proportion, ratio, and/or amount in the generalpopulation or a population of subjects who do not have dysbiosis, aninflammatory disease, or a gastrointestinal infection.

Embodiment 101

The method of any one of embodiments 85 to 100, further comprisingdirecting the subject to receive treatment or further testing ormonitoring for dysbiosis or an inflammatory disease if the biologicalsample is detected to be pro-inflammatory.

Embodiment 102

The method of any one of embodiments 85 to 101, further comprisingadministering the composition of any one of embodiments 52 to 62 to thesubject if the biological sample is detected to be pro-inflammatory.

Embodiment 103

The method of any one of embodiments 95 to 102, wherein the subjectcomprises a gastrointestinal microbiome that

-   -   (a) has an increased proportion of Streptococcus spp.,        Bifidobacterium spp., and Enterococcus spp. compared to a        healthy or general population;    -   (b) has a reduced proportion of Alternaria alternata,        Aspergillus flavus, Aspergillus cibarius, and Candida sojae        compared to a healthy or general population;    -   (c) has an increased proportion of Candida albicans and        Debaryomyces spp. compared to a healthy or general population;    -   (d) has a reduced proportion of Bifidobacteria spp.,        Lactobacillus spp., Faecalibacterium spp. and Akkermansia spp.        compared to a healthy or general population;    -   (e) has a reduced proportion of Malassezia spp. compared to a        healthy or general population    -   (f) has an increased proportion of Bacterioides spp.,        Ruminococcus spp., Prevotella spp., or Bifidobacterium spp.        compared to a healthy or general population; or    -   (g) has an increased proportion of Enterococcus faecalis,        Enterococcus faecium, or Clostridium difficile compared to a        healthy or general population.

Embodiment 104

The method of any one of embodiments 63 to 103, further comprisingdetermining whether the subject has a gastrointestinal microbiome that

-   -   (a) has an increased proportion of Streptococcus spp.,        Bifidobacterium spp., and Enterococcus spp. compared to a        healthy or general population;    -   (b) has a reduced proportion of Alternaria alternata,        Aspergillus flavus, Aspergillus cibarius, and Candida sojae        compared to a healthy or general population;    -   (c) has an increased proportion of Candida albicans and        Debaryomyces spp. compared to a healthy or general population;    -   (d) has a reduced proportion of Bifidobacteria spp.,        Lactobacillus spp., Faecalibacterium spp. and Akkermansia spp.        compared to a healthy or general population;    -   (e) has a reduced proportion of Malassezia spp. compared to a        healthy or general population    -   (f) has an increased proportion of Bacterioides spp.,        Ruminococcus spp., Prevotella spp., or Bifidobacterium spp.        compared to a healthy or general population; or    -   (g) has an increased proportion of Enterococcus faecalis,        Enterococcus faecium, or Clostridium difficile compared to a        healthy or general population.

Embodiment 105

A method of treating or preventing dysbiosis or an inflammatory diseasein a subject determined to have or be at risk of developing dysbiosis oran inflammatory disease according to the method of any one ofembodiments 85 to 103, comprising administering a treatment fordysbiosis or the inflammatory disease to the subject.

Embodiment 106

A method of monitoring the effect of treatment for dysbiosis or aninflammatory disease, the method comprising: (i) obtaining a biologicalsample from the subject; and (ii) detecting whether the biologicalsample is pro-inflammatory.

Embodiment 107

A method of determining an inflammatory disease activity in a subject,the method comprising: (i) obtaining a biological sample from thesubject; and (ii) detecting whether the biological sample ispro-inflammatory.

Embodiment 108

A method of detecting an anti-inflammatory metabolite in a subject thathas or is at risk for developing an inflammatory disease, said methodcomprising: (i) obtaining a biological sample from said subject; and(ii) determining an expression level of an anti-inflammatory metabolitein said biological sample.

Embodiment 109

A method of determining whether a subject has or is at risk ofdeveloping dysbiosis or an inflammatory disease, the method comprising:(i) detecting an expression level of one or more anti-inflammatorymetabolites or pro-inflammatory metabolites in a subject; (ii)determining whether the expression level is increased or decreasedrelative to a standard control, wherein an elevated expression level ofan pro-inflammatory metabolite or a decreased expression level of ananti-inflammatory metabolite relative to the standard control indicatesthat the subject has or is at risk of developing an inflammatorydisease; and (iii) based at least in part on the expression level instep (ii), determining whether the subject has or is at risk fordeveloping an inflammatory disease.

Embodiment 110

A method of monitoring the effect of treatment for an inflammatorydisease in a subject undergoing inflammatory disease therapy or apatient that has received inflammatory disease therapy comprising: (i)determining a first expression level of an anti-inflammatory orpro-inflammatory metabolite in the subject at a first time point; (ii)determining a second expression level of an anti-inflammatory orpro-inflammatory metabolite in the subject at a second time point; and(iii) comparing the second expression level of an anti-inflammatory orpro-inflammatory metabolite to the first expression level of ananti-inflammatory or pro-inflammatory metabolite, thereby determiningthe effect of treatment for an inflammatory disease in the subject.

Embodiment 111

A method of determining an inflammatory disease activity in a subject,the method comprising: (i) detecting an expression level of one or moreanti-inflammatory or pro-inflammatory metabolites in a subject; (ii)determining whether the expression level is modulated relative to astandard control, thereby determining an inflammatory disease activityin the subject; and (iii) based at least in part on the expression levelin step (ii), determining the inflammatory disease activity in thesubject.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1. Rationally Designed Microbial Consortium for GastrointestinalMicrobiome Restitution

Without being bound by any scientific theory, Lactobacillus johnsoniishifts the composition of the gut microbiome and increases specificanti-inflammatory fatty acids and carbohydrate metabolites in thegastrointestinal tract. Although some beneficial metabolites arepredicted to be microbially produced (e.g., by L. johnsonii and thebacterial species it co-enriches within the gut microbiome), it is alsolikely that others are host derived in response to an altered gutmicrobiome. In a study of neonates supplemented daily for the first sixmonths of age with Lactobacillus rhamnosus GG, an altered gut microbiomeassociated with similar metabolic enrichments persisted for up to 12months after the cessation of supplementation with Lactobacillus.

Surprisingly, a bacterial population comprising a consortium ofbacterial species may be used to prevent or treat chronic inflammatorydisease by introducing or restoring the metabolic capacity to regulateinflammatory responses. The consortium of bacterial species (the“consortium”) achieves this by altering microbial colonization patternsin the gastrointestinal tract, and, most importantly, introducing orrestoring the capacity to produce a suite of anti-inflammatorymetabolites necessary for down-regulation of pro-inflammatory responses.Much of the risk of childhood disease is associated with early lifeevents in microbiological development and this consortium offers theopportunity to treat high-risk neonates and infants.

The consortium may be used as a therapeutic grade formulation orover-the-counter supplement to, e.g., direct appropriate neonatal gutmicrobiome development and immune maturation. The consortium may also beused as a replacement for fecal transplantation for chronic inflammatorydiseases in which member of the consortium are characteristicallydepleted or as a supplement to direct gut microbiome re-developmentfollowing perturbation (e.g., peri- or post-antibiotic or anti-microbialadministration).

Without being bound by any scientific theory, the species in theexemplary consortium work together with the main anchor probioticspecies, L. johnsonii, in a symbiotic manner, with each providing othermembers of this bacterial guild with nutrients and co-factors for theirsurvival and modulation of host immunity. Intervention using a regimenof microbial consortium (Lactobacillus johnsonii. Akkermansiamuciniphila, Faecalibacterium prausnitzii and Myxococcus xanthus),provides improved protection against allergic sensitization due to aneffect that is greater than the sum of the effects of the individualconsortium members when administered alone. Using a similar mouse modelto that previously published (Fujimura et al., (2014). Proc. Natl. Acul.Sci. 111(2) 805-810), an allergic challenge was combined withsupplementation of the exemplary microbial consortium. Set forth herein,the host immune response and allergic response was evaluated usinghistology, qRT-PCR, and flow cytometry.

Cockroach Allergen (CRA) Murine Model.

To investigate the protective effects of supplementation, C57BL/6 mice(7-8 weeks old) were intratracheally sensitized (Day 1-3) andsubsequently challenged once a week with cockroach allergen (CRA) for atotal of three weeks. The mice were concurrently supplemented withphosphate buffered saline (PBS, negative vehicle control), L. johnsonii(Lj), the microbial consortium lacking L. johnsonii (C-Lj), a completeconsortium (C+Lj), or a heat killed complete consortium (C+Lj HeatKilled, control for metabolically inactive consortium). In the firstweek supplementation was performed daily, followed by supplementationtwice a week for the remaining two weeks. All supplementations wereperformed by oral gavage using bacteria resuspended in 100 μl of PBS. Atthe conclusion of the study, mice were euthanized, and various tissues(lung, spleen, ileum) were collected for downstream analyses.

Lung Histology.

Lung tissue was collected from each animal and immediately fixed inCarnoy's solution overnight and subsequently dehydrated in 70% ethanol.Three samples from each group were randomly chosen for embedding inparaffin and staining with hematoxylin and eosin (H&E) or Periodicacid-Schiff (PAS). Images for each stained sample were captured using anAperio Scanscope XT (Leica Biosystems) at 20× magnification. ImageJ wasalso used to quantify the amount of mucin staining represented in eachPAS-stained slide using set threshold parameters in the RGB stack basedon the green channel. The percentage of the image that fell within thethreshold values was measured and represented the percentage of positivestaining within each image analyzed.

qRT-PCR for Gene Expression.

The mRNA from mouse lung was extracted using an AllPrep DNA/RNA Mini Kit(Qiagen). Prior to RNA isolation, lung samples were placed in LysingMatrix A tubes (MP Bio) with 600 μl of Buffer RLT. Samples werebead-beaten using MPBio FastPrep-24 homogenizer at 5.5 m/s for 30 s.Manufacturer's instructions were followed for the remainder of the RNAisolation procedure. A total of 1.0 μg of RNA per sample was DNasetreated and reverse-transcribed using the RT2 First Strand Kit (Qiagen)per the manufacturer's instructions. Quantitative PCR for allergyassociated gene expression was performed using the Custom RT ProfilerPCR Array (Qiagen) on a QuantStudio 6 Flex System. Reaction conditionswere as follows: 95° C. for 10 min, followed by 40 cycles of 95° C. for15 s and 60° C. for 1 min. Gene expression of cytokines was normalizedto GAPDH and expressed as fold change compared to gene expression inCRA-challenged PBS-vehicle gavaged mice. Statistical analysis ofcytokine expression levels was preformed using Prism 6 software. Geneexpression between experimental groups was compared using a Mann-WhitneyU test, with p-values≤0.05 considered significant.

CD4⁺ T Cell Isolation and Flow Cytometry Analysis.

Mouse spleens were removed and placed in ice-cold R10 media (RPMI 1640supplemented with 10% heat-inactivated FCS, 2 mM L-glutamine, and 100U/ml penicillin-streptomycin) (Life Technologies, Carlsbad, Calif.).Tissues were mechanically homogenized using sterile scalpels, followedby collagenase digestion (C6885, Sigma, 1 mg/ml) at 37° C. for 30minutes in 1:1 R10-PBS solution. The single cell suspensions wereobtained by passing digests 10× through a 16-gauge, blunt-end cannulafollowed by filtrations through a 40 μm filter. Cell suspensions werewashed twice with ice cold PBS (2% FCS, 2 mM EDTA) and centrifuged at1,200 rpm, 4° C., for 10 min to pellet, and resuspended in R10-EDTAmedia (R10 with 2 mM EDTA) on ice. One million cells were dispensed intoeach tube for subsequent antibody staining and analysis. Single-cellsuspensions of splenocytes from each mouse were aliquoted (1 millioncells per well) and subsequently stained with antibodies CD4 (RM4-5, BDBiosciences, Franklin Lakes, N.J.), CD8a(53-6.7, BD), CXCR5(SPRCL5,eBioscience, San Diego, Calif.), PD-1(RMP1-30, BioLegend, San Diego,Calif.), CD25(PC61, BD), and live/dead aqua stain (Life Technologies).Following surface staining, cells were permeabilized using BDCytofix/Cytoperm and incubated with CD3e(500A2, BD), IFNγ(XMG1.2, BD),IL-4(11B11, BD), IL-17 DEC(eBiol7B7, eBioscience), and FoxP3(FJK-16s,eBioscience) specific antibodies for internal staining. Stained cellswere assayed via flow cytometry on a BD LSR II (BD Biosciences).

Statistical Analysis.

Statistical analyses were performed using GraphPad Prism 6 software.Experimental groups were compared by a Kruskal-Wallis test with a Dun'smultiple comparison post-test to determine if there were any significantdifferences between sample groups. In addition, Mann-Whitney tests wereused in some cases to directly compare two groups of values.P-values≤0.05 were considered significant.

Results.

Supplementation with the complete consortium (C+Lj) provides the mostrobust protection against allergic sensitization. Protection isassociated with significant decreases in lung mucin secretion (FIGS.1A-1B), Muc5 gene expression (FIG. 2), and in Th2 cytokine expression(FIGS. 3A-3C). Protection against allergic sensitization by C+Lj iscorrelated with systemic increases in IL-17 secreting T helper cells(FIG. 4). L. johnsonii is more effective than L. rhamnosus GG andnecessary for the attenuation of allergic sensitization associatedMuc5ac expression in the lung of CRA challenged C57BL/6 mice (FIG.5A-5B). The gut microbiota forms a complex functional network thatinfluences both individual microbial members and host immune responses.Rationally designed microbial gastrointestinal consortium providegreater attenuation of allergic airway sensitization than an individualprobiotic species.

Example 2. Effects of Consortium Supplementation on a Murine Model ofAirway Allergic Sensitization

Without being bound by any scientific theory, the therapeutic consortium(TC) represents a seed microbial guild that aids in the development of ahealthy human gut microbiome. A study in C57BL/6 mice was designed,which have a distinct gut microbiome from BALB/c animals and are notprotected against allergic airway sensitization followingsupplementation with L. johnsonii alone, to determine the effects of TCsupplementation on allergic airway sensitization.

To investigate the protective effects of TC supplementation C57BL/6 micewere intratracheally sensitized (days 0-2) and subsequently challengedwith cockroach allergen (CRA) on days 14 and 20 over the course of athree week period (FIG. 10). The mice were supplemented with eitherphosphate buffered saline (PBS, negative vehicle control) or the TC ondays 0-5, 8, 12, 16, and 19 via oral gavage (FIG. 10). Table 2 and FIG.17 show treatment groups utilized in this study.

Applicants examined the microbial community composition in the feces ofanimals in different treatment groups using 16S rRNA sequencing. Thecommunity present in that of the TC-supplemented animals wassignificantly compositionally distinct from that of the control groups(FIG. 11A). Importantly, the TC-supplemented group was enriched forspecies with the potential for immunomodulatory activity. For example,Bifidobacterium and specific Clostridia species belonging to Clade IVand XIV have been shown to induce T-regulatory cells. In addition,Lachnospira species have been identified as protective against allergicsensitization disease development. Expansion of Bacteroides wascharacteristic of allergic sensitization in control animals. Inconclusion, oral supplementation of mice with the TC promotes increasedrelative abundance of genera associated with induction of immunetolerance (e.g., Bifidobacteria, Clostridia, Lachnospira andRuminococcus; FIGS. 11A and 11B).

Oral supplementation with the TC promoted metabolic reprogramming inboth the gut lumen and periphery (FIGS. 12A-12B and FIGS. 18A-18C).Increased levels of itaconate, which is associated with a repairmacrophage effector phenotype, were also identified in TC supplementedanimals.

TC supplemented mice demonstrated significantly reduced allergicinflammation in response to CRA challenge compared with CRA challengedanimals treated with PBS (FIGS. 13A-13B; FIGS. 14A-14B; FIGS. 15A-15C).Thus, TC supplementation significantly reduced allergic inflammation ina murine model of airway allergic sensitization.

Oral supplementation of mice with the TC resulted in a repair macrophageeffector phenotype (FIGS. 16A-16F). Therefore, TC supplementation iscapable of initiating a repair macrophage effector phenotype in a murinemodel of airway allergic sensitization.

Example 3. In Vitro Assay for Assessment of Immune Activation StatusUsing Human Fecal Water or Microbial Products

One of the shortcomings of human microbiome studies is the lack ofparallel objective immune status information. Provided herein arepartner assays for human microbiome studies to determine the extent ofimmune activation associated with a variety of bodily fluids, such asfecal water or broncheoalveolar lavage fluid, or to assess the capacityof microbial species, or combinations of microbial species to induceimmune activation or, conversely induce immune tolerance. The assaysprovided herein may be used as diagnostics for chronic inflammatorydiseases, as well as for screening for bioactive microbial products thatinduce immune phenotypes associated with disease (and by extensionidentify target pathways for therapeutic intervention) or representnovel microbial biotherapeutics. No known assay to date has thiscapacity.

Fecal samples (250 mg) were added to warm PBS (250 μl, containing 20%FCS) at 1 g/1 ml, (w/v), followed with vigorous vortex for 1 minute.Fecal mixtures were incubated for 10 minutes in 37° C., prior to removalof cellular material by microcentrifugation at 14,000 rpm for 5 minutes.Resulting fecal water was sterilized through a 0.2 μm filter and used inDC co-incubations.

Peripheral blood mononuclear cells (PBMCs) were isolated from peripheralblood of healthy adult donors by Ficoll-Hypaque gradient centrifugation.DCs were first enriched from the PBMCs using the EasySep™ Human Pan-DCPre-Enrichment Kit (STEMCELL Technologies, Vancouver, Canada). EnrichedDCs (0.5×10⁶ cells/ml) were co-incubated for 48 hours with fecal water(25 μl) and cultured in 96-well plates, in R10 media (RPMI 1640 with 10%heat-inactivated FCS with 2 mM L-glutamine and 100 U/mlpenicillin-streptomycin; Life Technologies, Carlsbad, Calif.)supplemented for the first 24 hours with 10 ng/ml GM-CSF and 20 ng/mlIL-4 for. A combination of DC growth factors (10 ng/ml TNF-α, 10 ng/mlIL-1β, 10 ng/ml IL-6, and 1 μM PGE2) were added to the culture for thesubsequent 24 hours of incubation. At the end of 48 hour treatment, DCswere washed (once) in fresh media prior to co-culture with CD4+lymphocytes.

Autologous CD4+ T lymphocytes were purified from PBMC's by negativeselection using a CD4+ T-cell isolation kit (Miltenyi Biotec, BergischGladbach, Germany). These isolated T cells were suspended in the TexMACSMedium (Miltenyi Biotec) prior to being added to fecal water exposed DCsat a ratio of 10:1 in the presence of soluble anti-CD28 and anti-CD49d(1 μg/ml). DC and T-cells were co-cultured for 120 hours and replenishedwith fresh TexMACS Medium every 48 hours. Cells were stimulated withPhorbol Myristate Acetate-lonomycin (Sigma) and GolgiPlug (BDBiosciences) for the final 16 hours of co-incubation. Cell-free mediafrom these co-cultures were collected and evaluated by ELISA (BioLegend)for human cytokines IL-4, IL-10, and IL-13 concentrations.

Single-cell suspensions were stained using two separate panels(phenotype panel and cyctokine panel) of antibodies including BDBiosciences Abs anti-CD3 (SP34-2), anti-CD4 (SK3), anti-CD25 (M-A251),anti-IFNγ (B27), anti-CD8a (RPA-T8, BioLegend), Miltenyi Biotec Absanti-IL-10 (JES3-9D7), anti-IL-4 (7A3-3), Affymatrix eBioscience Absanti-IL-22 (22URTI), anti-IL-17A (64DEC17) and anti-FoxP3 (PCH101). Deadcells were identified using LIVE/DEAD® Aqua Dead Cell Stain (LifeTechnologies). Cells were permeablized by either Cytofix/Cytoperm™ (BDBioscience) or Fixation/Permeabilization (Affymatrix eBioscience) tostain for intracellular markers, IFNγ, IL-4, IL17A, IL-22, IL-10, FoxP3.Upon flow analysis, live T cells were gated as CD3+CD4+ cells. Amongstthe CD4+ T cell sub-populations, Th1 were IFNγ+, Th2 were IL-4+, Th17cells were IL-17A+, Th22 were IL17A-negative and IL-22+, andT-regulatory cells were both CD25^(hi) and FoxP3^(hi). Stained cellswere assayed via flow cytometer on a BD LSR II (BD Biosciences).

Fecal water from a non-atopic neonate significantly reduces CD4+ IL4 andIL13 expression in vitro. Atopy is associated with early-lifegastrointestinal bacterial overgrowth and murine microbial metabolism,thus implicating the neonatal gut microbiome in allergic diseasedevelopment. Microbiota analysis of 298 early-life stool samples from abirth cohort revealed the existence of three compositionally distinctNeonatal Gut Microbiotypes (NGM1, NGM2 and NGM3). NGM3 neonatesexhibited significantly higher relative risk for predominantlymultisensitized atopy at age two (p<0.03) compared with NGM1 (RR=2.94;95% CI 1.42-6.09) or NGM 2 (RR=2.06; 95% CI 1.01-4.19), and were morelikely to report doctor-diagnosed asthma (p<0.03). Lower-risk NGMs weresignificantly enriched for commensal bacteria, fungi and a range ofluminal anti-inflammatory lipids and carbohydrates. NGM3 neonatesexhibited commensal microbial depletion, fungal expansion and metabolicreprogramming manifest as increased pro-inflammatory lipids andhost-derived sterols associated with fungal infection.

Findings from this study of neonatal and infant gut microbiomesindicated that neonatal metabolic reprogramming was associated with riskof atopy development at age two and that neonates that exhibitedsignificant increases in anti-inflammatory carbohydrates and lipids intheir luminal contents were at significantly decreased risk for allergicsensitization. Previous murine studies have indicated thatmicrobial-derived short chain fatty acids afford protection againstairway allergen challenge. Applicants rationalized that fecal water fromlow-risk neonates (NGM1) which was enriched for known anti-inflammatorylipids and carbohydrates would exhibit the capacity to reduceallergy-associated cytokine expression. Applicants therefore incubatedfilter-sterilized fecal water from an NGM1 neonate with peripheral bloodmononuclear cell-derived dendritic cells isolated from two distincthealthy adult donors, prior to their co-incubation with autologouslypurified naïve T-cells followed by ionomycin stimulation. Flow cytometryand ELISA analyses, used to examine T-helper 2 (CD4+, IL4+) cells andcytokine production respectively, indicated that fecal water did notsubstantially influence the number of Th2 cells (FIG. 6A), butsignificantly and consistently suppressed pro-inflammatory IL4 and IL13expression (p<0.01 for both; FIG. 6B and FIG. 6C) across both donors.

In vitro DC-T-cell activity assay permits identification of microbeswith pro-inflammatory potential. In our studies of the neonatal gutmicrobiome and atopy, Candida enrichment and host responses to fungalinfection (beta-sitosterol and stigmasterol), were amongst the featuresthat characterized high-risk for atopy NGM3 participants. Murine studiesemploying antimicrobial ablation of the commensal gastrointestinalmicrobiome, followed by instillation of Candida albicans spores, havepreviously demonstrated enhanced allergic sensitization even in theabsence of allergen exposure. Without wishing to be bound by anyscientific theory, it was rationalized that Candida enrichment in thegut microbiome of NMG3 neonates may promote adaptive T-helper cellsubsets associated with atopy. Using Candida-selective Sabouraud media,four distinct species were isolated from NGM3 neonatal stool samples andidentified using full length ITS sequencing as C. metapsilosis, C.parapsilosis, C. orthopsilosis, and C. tropicalis. Filter-sterilizedcell-free supernatant (CFS) from cultures of these four Candida specieswas used to stimulate peripheral blood mononuclear cell-deriveddendritic cells prior to their co-incubation with naïve T-cells in thepresence or absence of cockroach antigenic stimulation. Flow cytometryanalysis was used to examine T-helper 2 (CD4+, IL4+) and T-regulatory(CD4+, IL10+) subsets. CFS from each of the gastrointestinal Candidaspecies induced significant increases in Th-2 cell proliferationcompared to the control (sterile culture medium) exposure, irrespectiveof cockroach allergen challenge (FIG. 7A). Significant increases werealso observed for other pro-inflammatory cytokine producing T-helpercell subsets (TH1, TH17 and TH22). However T-regulatory subsets did notexhibit a consistent significant increase in numbers, indeed mostspecies did not affect T-reg numbers, with one, C. tropicalis, inducingT-regs and another C. metapsilosis inducing a significant reduction inT-reg cell numbers, only in the presence of cockroach allergenstimulation (FIG. 7B). Hence these in vitro analyses corroborateprevious animal studies and indicate that the secreted products ofdistinct Candida species enriched in the gut microbiome of neonates withsignificantly higher relative risk for atopy at age two have thecapacity to drive Th-2 proliferation and cytokine secretion irrespectiveof antigen presentation by DCs.

In vitro DC/T-cell fecal water assay can be used to discriminatesub-sets of ulcerative colitis patients that exhibit distinct fecalmicrobiotypes and differ significantly in disease severity. Statisticalanalyses has permitted us to identify three sub-groups of UC patientsbased on microbiota composition (bacterial and fungal profiling),referred to MBT-1 to -3). The clinical relevance of gut microbiota-basedstratification of our UC patients was assessed by an inter-microbiotypecomparison of disease severity (Simple Clinical Colitis Activity (SCCA)index duration (number of years since UC diagnosis), extracolonicmanifestations (arthritis, pyoderma gangrenosum, erythema nodosum, anduveitis) and number of first- and second-degree relatives with IBD.MBT-1 patients exhibited higher median SCCA score compared to MBT-2 andMBT-3 groups (FIG. 8A). These patients also exhibited more extracolonicmanifestations, and trended towards longer disease duration and agreater number of first- and second-degree relatives diagnosed with IBD(FIGS. 8B-8D).

In an effort to determine the capacity of healthy and disease-associatedmicrobiota to influence adaptive immune responses, we next developed anin vitro assay involving exposure of dendritic cells (DCs; obtained fromhealthy human donors) to filter-sterilized fecal water from participantsin our study, prior to co-culture of exposed DCs with naïve T-cellsobtained from the same donor. Flow cytometry was used assess resultingT-cell populations and phenotypes. Compared to healthy control subjects,UC patients were characterized by significant differences in the ratioof CD4+Th1 and Th2 cells; patients exhibited significantly decreasedTh1:Th2 ratio (FIG. 9A). Other CD4+ populations (Th17, Th22, andT-regulatory cell) abundances did not exhibit significant differences inrelative numbers, nor did CD8+ cell numbers differentiate based onhealth status. While differences in the Th1:Th2 ratio existed acrosshealthy subjects and UC patients, we postulated that UC-associated gutmicrobiotypes associated with significantly different disease severityscores would also exhibit concomitant differences in this ratio. Wetherefore examined cytokine production patterns based onUC-microbiotype. The MBT-1 group, which exhibited the highest diseaseseverity scores exhibited a significantly reduced Th1:Th2 ratio (FIG.9B), which was associated with an expansion of CD4+, IL4 expressingT-cells (FIG. 9C). Other CD4+ T-cell populations (Th17, Th22, and Treg)also trended towards expansion in MBT-1 group, compared to the MBT2 andMBT3 groups. Additionally, the MBT-1 group exhibited a significantincrease in IL-17 producing CD8+ T-cells compared to either MBT-2 andMBT-3 patient samples (FIG. 9D). These in vitro data are consistent withclinical observations in that compared to healthy subjects, UC patientsare significantly skewed towards a Th2-enriched population of CD4+cells, and that UC-microbiotypes exhibit significant differences in boththeir degree of Th2 skewing and expansion of IL17 producing cytotoxicCD8+ cells. Hence microbiological stratification allows identificationof immunologically distinct UC patient populations.

TABLE 2 Treatment groups utilized in murine model of airway allergicsensitization study. Gavage Cockroach Allergen (CRA) Intervention Group−(PBS) PBS Vehicle No CRA + PBS Vehicle CRA + PBS + TherapeuticConsortium CRA + TC (TC) + L. johnsonii (Lj) CRA + Lj + Consortiumwithout Lj (C) CRA + C + Heat-Killed TC (HKTC) CRA + HKTC

TABLE 3 Supplementation with Therapeutic Consortium results in metabolicreprogramming leading to an increase in specific lipid compounds.Compound Compound Type Subtype Compound Lipids Phospholipids1-palmitoyl-2gamma-linolenoyl- GPC (16:0/18:3n6) Lipids PhospholipidsOleoylcholine Lipids Phospholipids 1-linoleoyl-2-arachidonoyl-GPC(18:2/20:4n6) Lipids Phospholipids 1-palmitoleoyl-2-linoleoyl-GPC(16:1/18:2) Lipids Phospholipids 1-palmitoyl-2-alpha-linolenoyl-GPC(16:0/18:3n3) Lipids Phospholipids 1,2-dioleoyl-GPE (18:1/18:1) LipidsPhospholipids 1-stearoyl-2-linoleoyl-GPE (18:0/18:2) LipidsPhospholipids 1-palmitoyl-2-arachidonoyl-GPE (16:0/20:4) LipidsPhospholipids 1-stearoyl-2-oleoyl-GPE (18:0/18:1) Lipids Phospholipids1-stearoyl-2-arachidonoyl-GPE (18:0/20:4) Lipids Phospholipids1-palmitoyl-2-palmitoleoyl-GPC (16:0/16:1) Lipids Phospholipids1-stearoyl-2-linoleoyl-GPC (18:0/18:2) Lipids Phospholipids1-palmitoyl-2-arachidonoyl-GPC (16:0/20:4) Lipids Phospholipids1,2-dioleoyl-GPC (18:1/18:1) Lipids Phospholipids1-stearoyl-2-oleoyl-GPC (18:0/18:1) Lipids Phospholipids1-stearoyl-2-arachidonoyl-GPC (18:0/20:4) Lipids Phospholipids1-palmitoyl-2-linoleoyl-GPC (16:0/18:2) Lipids Phospholipids1-palmitoyl-2-oleoyl-GPC (16:0/18:1) Lipids Phospholipids1,2-dipalmitoyl-GPC (16:0/16:0) Lipids Plasmalogens1-(1-enyl-oleoyl)-GPE (P-18:1) Lipids Plasmalogens1-(1-enyl-palmitoyl)-GPE (P-16:0) Lipids Plasmalogens1-(1-enyl-palmitoyl)-2-linoleoyl- GPC (P-16:0/18:2) Lipids Plasmalogens1-(1-enyl-palmitoyl)-2- arachidonoyl-GPC (P-16:0/20:4) LipidsPlasmalogens 1-(1-enyl-palmitoyl)-2-oleoyl-GPC (P-16:0/18:1) LipidsPlasmalogens 1-(1-enyl-palmitoyl)-2- arachidonoyl-GPE (P-16:0/20:4)Lipids Plasmalogens 1-(1-enyl-palmitoyl)-2-linoleoyl- GPE (P-16:0/18:2)Lipids Plasmalogens 1-(1-enyl-palmitoyl)-2-oleoyl-GPE (P-16:0/18:1)

TABLE 4 Supplementation with Therapeutic Consortium results in metabolicreprogramming leading to a decrease in specific carbohydrate, lipid,energy compounds. Compound Type Compound Subtype Compound CarbohydratesGlycolysis/Pyruvate 1,5-anhydroglucitol CarbohydratesGlycolysis/Pyruvate Glucose Carbohydrates Glycolysis/Pyruvate PyruvateCarbohydrates Glycolysis/Pyruvate Glycerate Carbohydrates Pentose RiboseCarbohydrates Pentose Ribitol Carbohydrates Pentose RibonateCarbohydrates Pentose Xylose Carbohydrates Pentose ArabinoseCarbohydrates Pentose Arabitol/Xylitol CarbohydratesFructose/Mannose/Galactose Mannitol/Sorbitol CarbohydratesFructose/Mannose/Galactose Mannose CarbohydratesFructose/Mannose/Galactose Galactitol (dulcitol) Lipids PolyunsaturatedFatty Acids Docosapentaenoate (n3 DPA; 22:5n3) Lipids PolyunsaturatedFatty Acids Adrenate (22:4n6) Lipids Polyunsaturated Fatty AcidsDocosadienoate (22:2n6) Lipids Polyunsaturated Fatty AcidsDihomo-linoleate (20:2n6) Lipids Acyl-glycerols 1-myristoylglycerol(14:0) Lipids Acyl-glycerols 2-myristoylglycerol (14:0) LipidsAcyl-glycerols 1-pentadecanoylglycerol (15:0) Lipids Acyl-glycerols1-palmitoylglycerol (16:0) Lipids Acyl-glycerols 2-palmitoylglycerol(16:0) Lipids Acyl-glycerols 1-margaroylglycerol (17:0) LipidsAcyl-glycerols 1-oleoylglycerol (18:1) Lipids Acyl-glycerols2-oleoylglycerol (18:1) Lipids Acyl-glycerols 1-linoleoylglycerol (18:2)Lipids Acyl-glycerols 2-linoleoylglycerol (18:2) Lipids Acyl-glycerols1-linolenoylglycerol (18:3) Lipids Acyl-glycerols1-docosahexaenoylglycerol (22:6) Lipids Acyl-glycerols2-docosahexaenoylglycerol (22:6) Lipids Acyl-glycerols1-palmitoleoylglycerol (16:1) Lipids Acyl-glycerols2-palmitoleoylglycerol (16:1) Lipids Acyl-glycerols1-eicosapentaenoylglycerol (20:5) Lipids Acyl-glycerols2-eicosapentaenoylglycerol (20:5) Lipids Branched Fatty Acids15-methylpalmitate Lipids Branched Fatty Acids 17-methylstearate LipidsBranched Fatty Acids 2-hydroxyglutarate Lipids Branched Fatty Acids1-dihomo-linoleoylglycerol (20:2) Lipids Long Chain Fatty AcidsPentadecanoate (15:0) Lipids Long Chain Fatty Acids Palmitate (16:0)Lipids Long Chain Fatty Acids Margarate (17:0) Lipids Long Chain FattyAcids 10-heptadecenoate (17:1n7) Lipids Long Chain Fatty Acids Stearate(18:0) Lipids Long Chain Fatty Acids Nonadecanoate (19:0) Lipids LongChain Fatty Acids 10-nonadecenoate (19:1n9)* Lipids Long Chain FattyAcids Arachidate (20:0) Lipids Long Chain Fatty Acids Eicosenoate (20:1)Lipids Long Chain Fatty Acids Erucate (22:1n9) Lipids Long Chain FattyAcids Oleate/Vaccenate (18:1) Energy TCA Cycle Citrate Energy TCA CycleSuccinate Energy TCA Cycle Mesaconate

Example 4. Neonatal Gut Microbiota Associates with ChildhoodMultisensitized Atopy and T Cell Differentiation

Gut microbiota bacterial depletions and altered metabolic activity at 3months are implicated in childhood atopy and asthma¹. We hypothesizedthat compositionally distinct human neonatal gut microbiota (NGM) exist,and are differentially related to relative risk (RR) of childhood atopyand asthma. Using stool samples (n=298; aged 1-11 months) from a USbirth cohort and 16S rRNA sequencing, neonates (median age, 35 d) weredivisible into three microbiota composition states (NGM1-3). Eachincurred a substantially different RR for multisensitized atopy at age 2years and doctor-diagnosed asthma at age 4 years. The highest riskgroup, labeled NGM3, showed lower relative abundance of certain bacteria(for example, Bifidobacterium, Akkermansia and Faecalibacterium), higherrelative abundance of particular fungi (Candida and Rhodotorula) and adistinct fecal metabolome enriched for pro-inflammatory metabolites. Exvivo culture of human adult peripheral T cells with sterile fecal waterfrom NGM3 subjects increased the proportion of CD4⁺ cells producinginterleukin (IL)-4 and reduced the relative abundance of CD4⁺CD25⁺FOXP3⁺ cells. 12,13-DiHOME, enriched in NGM3 versus lower-risk NGMstates, recapitulated the effect of NGM3 fecal water on relative CD4CD25forkhead box P3 (FOXP3)⁺ cell abundance. These findings suggest thatneonatal gut microbiome dysbiosis might promote CD4⁺ T cell dysfunctionassociated with childhood atopy.

Atopy, the propensity to produce IgE antibodies in response toallergens, is one of the most common chronic health issues² and isconsidered to be a substantial risk factor for childhood asthmadevelopment³. Recently, the condition has been linked to bacterialtaxonomic depletions in the human gut microbiota at 3 months, but not at12 months, of age¹. We therefore hypothesized that compositionally andfunctionally distinct neonatal (˜1 month of age) gut microbiota statesexist, and that their associated products idiosyncratically influenceCD4⁺ populations in a manner that relates to the RR of atopy and asthmadevelopment in childhood. We studied independent fecal samples collectedduring a study visit that targeted 1 month olds (median age 35 d; range16-138 d; n=130; ‘neonates’) or 6 month olds (median age 201 d; range170-322 d; n=168; ‘infants’) from participants in the racially andsocioeconomically diverse Wayne County Health, Environment, Allergy andAsthma Longitudinal Study birth cohort⁴. Predominantly multisensitizedatopy (PM atopy) at age 2 years was defined using latent-class analysis,an unsupervised statistical algorithm that clusters subjects accordingto their pattern of serum specific-IgE (sIgE) responses to a panel often food and aeroallergens⁵ (FIG. 26).

At the population level (independently of atopy status), bacterialcommunity α-diversity (taxon number and distribution) expanded withincreasing age (Pearson's correlation, r=0.47, P<0.001). In parallel,fungal α-diversity contracted (Pearson's correlation, r=−0.23,P=0.0014), and a reciprocal relationship between these microbialkingdoms existed (Shannon's index; Pearson's correlation, r=−0.24,P<0.001; FIG. 19). Both bacterial and fungal 3-diversity (interpersonaltaxonomic composition) were related to participant age (PERMANOVA;R²=0.056, P<0.001; and R²=0.034, P<0.001, respectively. Neonatal fecalmicrobiota were typically dominated by Bifidobacteriaceae,Enterobacteriaceae, Malasseziales (Malassezia) and Saccharomycetales(Saccharomyces). Infant participants exhibited sustained presence, butdiminished relative abundance, of Bifidobacteriaceae andEnterobacteriaceae, an expansion of Lachnospiraceae (Blautia andRuminococcus) and fungal communities characteristically dominated bySaccharomycetales (Saccharomyces and Candida), the dominant fungal orderin healthy adults⁶. These findings indicate an interkingdom gutmicrobial co-evolution along an age-associated developmental gradientover the first year of life.

To address our primary hypothesis, a Dirichlet multinomial mixture (DMM)model was used to group participants on the basis of bacterial-communitycomposition⁷; three distinct NGM states (NGM1, 2 and 3) represented thebest model fit (FIG. 23). PERMANOVA confirmed that NGM designationexplained a small but nontrivial proportion of bacterial β-diversity(PERMANOVA; R²=0.09, P<0.001), indicating that NGMs [which did notdiffer in age (Kruskal-Wallis; P=0.256; FIG. 20A)] may represent agradient of microbiota configurations in early-life. NGMs trended towardhaving a significant relationship with fungal β-diversity (Bray-Curtis;PERMANOVA, R²=0.037, P=0.068), signifying that each NGM co-associateswith a mycobiota that varies primarily in the relative abundance of thedominant fungal taxa present. Infant samples were divisible into twocompositionally distinct gut microbiota states, IGM1 (typicallyBifidobacteriaceae dominated) and IGM2 (typically Lachnospiraceaedominated (unweighted UniFrac; PERMANOVA, R²=0.032, P=0.001)), whichdiffered in age (Wilcoxon rank-sum, P=0.0257); IGM1 participants wereyounger. IGM states were not related to fungal community β-diversity(Bray-Curtis; PERMANOVA, R²=0.011, P=0.33), presumably because infantsubjects were consistently enriched for Saccharomycetales.

According to the conventional definition of atopy (IgE>0.35 IU/ml), nosignificant difference in RR between NGM groups was observed (FIG. 21).However, when the asthma-predictive⁵ PM atopy definition was used, NGM3participants incurred a higher RR of atopy at age 2 years, as comparedto either NGM1 (RR=2.94, 95% CI 1.42-6.09 P=0.004; FIG. 21) or NGM2groups (RR=2.06; 95% CI 1.01-4.19, P=0.048; FIG. 21). Even larger effectsizes for NGM3 were observed for RR of parental-reported,doctor-diagnosed asthma at age 4 years (FIG. 21). NGM-associated RR ofPM atopy was supported by the sum of specific IgE responses at age 2years (FIG. 20B). IGM participants did not exhibit different RRs for PMatopy (RR=1.02; 95% CI 0.59-1.75, P=0.94; FIG. 27) or asthma (RR=0.51;95% CI 0.22-1.17, P=0.11), possibly due to increased age range andmicrobial heterogeneity within this group. Using available early-lifecharacteristics, we identified factors including season of birth, age atsample collection and breastfeeding to be substantially distinct acrossIGM states. Detectable dog allergen (Can f 1) concentrations (P=0.045)in the home during the neonatal study visit (lowest in the NGM3 group)and baby gender (NGM3 was almost entirely male) significantly differedacross NGMs (P=0.038). Despite adjustment for these and other early-lifefactors commonly related to allergic disease, the relationship betweenNGM and atopy or asthma persisted. Only one other large pediatricgut-microbiota atopy study exists¹, the youngest participants of whichwere substantially older (˜100 d) than the neonates in our cohort(median age, 35 d). The application of our DMM model parameters to thisdata set identified two compositionally distinct groups(Bifidobacteria-dominated NGM1 and Lachnospiraceae-dominated IGM2;indicating that examination of neonatal stool samples is necessary toidentify distinct pioneer microbiota related to differential RR.

NGM3 participants were characteristically depleted of bacterial taxa,including Bifidobacteria (Bifidobacteriaceae), Lactobacillus(Lactobacillaceae), Faecalibacterium (Clostridiaceae) and Akkermansia(Verrucomicrobiaceae), when compared with the NGM1 group (zero-inflatednegative binomial regression (ZINB), Benjamini-Hochberg, q<0.05). Theseobservations were consistent when NGM3 was compared to NGM2 and alsowith previously described atopy-associated taxonomic depletions¹.Mycologically, NGM3 subjects were consistently depleted of multipleMalassezia taxa (ZINB, Benjamini-Hochberg, q<0.20, FIG. 28 and FIG.29)-striking, given our population-based observation that this genus ischaracteristically enriched in the neonatal gut microbiota. Fungaltaxonomic enrichments in the NGM3 group were also consistent whencompared to either of the lower-risk groups, and included Rhodotorulaand Candida (FIG. 28 and FIG. 29). Hence, neonatal interkingdommicrobiota dysbiosis is characteristic of PM atopy and asthmadevelopment in childhood.

NGM3-associated bacterial taxonomic alterations were predicted⁸ toresult in a deficiency in amino acid, lipid and xenobiotic metabolismpathways. Untargeted liquid chromatography mass spectrometry identifiedfecal metabolites present in a subset (n=28) of the representativesubjects from each NGM (those with the highest posterior probability ofNGM membership). Substantial correlations existed between the 16S rRNAprofile, predicted metagenome and the metabolome of NGMs (Procrustes;FIG. 30), indicating a deterministic relationship between bacterialcommunity composition and the metabolic microenvironment of the neonatalgut. Between-group comparisons identified specific metabolites enrichedin each NGM (Welch's t test; P<0.05). As previously reported fromanalysis of the urine of subjects with atopy¹, NGM3 participantsexhibited fecal enrichment of primary and secondary bile metabolites.However, more expansive metabolic dysfunction, involving lipid, aminoacid, carbohydrate, peptide, xenobiotic, nucleotide, vitamin and energymetabolism pathways—essentially, the bacterial pathways predicted to bedeficient in NGM3—was evident. Although the NGM1 and NGM2 groupsexhibited distinct metabolic programs, a common subset of metabolitesdifferentiated them from NGM3. These included anti-inflammatorypolyunsaturated fatty acids, docosapentaenoate (n3 DPA; 22 n5) anddihomo-γ-linolenate^(9,10) (DGLA; 20:3n3 or n6), succinate and thebreast-milk oligosaccharides, 3-fucosyllactose and lacto-N-fucopentaoseII, which are known to influence gut epithelial colonization^(11,12). Bycontrast, NGM3 participants were consistently enriched for 12,13-DiHOME,stigma- and sitosterols, 8-hydroxyoctanoate, α-CEHC and γ-tocopherol.

Sterile fecal water from NGM3 participants (compared to that from NGM1),decreased the ratio of CD4⁺IFNγ⁺:CD4⁺IL-4⁺ cells (linear mixed-effectsmodel (LME), P=0.095; FIG. 24), increased the proportion of CD4⁺IL-4⁺cells (LME, P<0.001; FIG. 22A) and the concentration of IL-4 released(LME, P=0.045; FIG. 22B) and reduced the percentage of CD4⁺CD25⁺FOXP3⁺cells (compared with control; LME, P<0.017; FIG. 22C) ex vivo,indicating that the NGM3 gut microenvironment promotes adaptive immunedysfunction associated with established atopic asthma. Weightedcorrelation network analysis identified 32 metabolic modules, one ofwhich discriminated the three NGMs (ANOVA; P=0.038; FIG. 22D) andcontained 12,13-DiHOME, which was identified both as a hub metabolite(highest module membership (MM) value=0.91; FIG. 22E) and mostNGM-discriminatory (highest MM to metabolite significance correlation(r=0.86, P<0.001; FIG. 22E). An observation supported by its relativeenrichment in NGM3 subjects compared to NGM1 and NGM2 (P<0.05 for both;FIG. 25). All concentrations of 12,13-DiHOME examined reduced theproportion of CD4⁺CD25⁺FOXP3⁺ cells, compared with vehicle treatment(LME, P=0.04, P<0.001, P=0.001 respectively; FIG. 22F).

These findings indicate that neonatal gut microbiota influencessusceptibility to childhood allergic asthma, potentially via alterationsin the gut microenvironment that influence CD4⁺ T cell populations andfunction. This suggests that very early-life interventions to manipulatethe composition and function of the gut microbiome might offer a viablestrategy for disease prevention.

Methods

Accession codes. All sequence data related to this study are availablefrom the European Nucleotide Archive (ENA) under accession numberPRJEB13896. Additional information is available in Fujimura et al. 2016.

Study Population. Pregnant women (n=1,258) between the ages of 21 and 49were recruited from August 2003-November 2007 as part of the WayneCounty Health, Environment, Allergy and Asthma Longitudinal Study(WHEALS). WHEALS is a prospective birth cohort from southeasternMichigan designed to investigate early-life risk factors for allergicdiseases, as previously described⁴. Briefly, women were consideredeligible if they lived in a predefined cluster of contiguous zip codesin and surrounding Detroit, Mich., had no intention of moving out of thearea and provided informed written consent. Five follow-up interviewswere conducted at 1, 6, 12, 24 and 48 months after the birth of theirchild, with the 24-month appointment being at a standardized studyclinic so that the child could be evaluated by a board-certifiedallergist. Stool samples were collected from the child at the 1- and6-month home visits. All aspects of this research were approved by theHenry Ford Hospital Institutional Review Board.

Sample criteria of WHEALS subjects for stool microbiome analyses. Forthis study, we selected children who had completed their 24-month clinicvisit, which included a blood draw for IgE measurements, and had haddust samples collected from their homes at the same time as theirstool-sample collection (in =308). Stool samples from children rangingfrom age 1-11 months were collected from field staff during home visitsand stored at −80° C. Samples were randomized before being shipped tothe University of California, San Francisco (UCSF), on dry ice, wherethey were also stored at −80° C. until processed.

PM-atopy and asthma definition. Blood drawn at the 2-year clinic visitwas used to determine participants' levels of total and tenallergen-specific IgEs (sIgE): Alternaria (Alternaria alternata), Germancockroach (Blattella germanica Bla g 2), dog (Canis lupus familiaris Canf 1), house dust mites (Dermatophagoides farinae Der f 1), hen's egg(egg), cat (Felis domesticus Fel d 1), cow's milk (milk), peanut(Arachis hypogaea), common ragweed (Ambrosia artemisiifolia) and Timothygrass (Phleum pratense). Specific IgEs were measured using the PharmaciaUniCAP system (ThermoFisher Scientific, Waltham, Mass., USA). Latentclass analysis was used to group participants into four discrete atopicclasses according to sensitization patterns of the ten allergen sIgEs,as with the entire WHEALS cohort⁵. Our subset was assigned to one offour latent classes: (i) Low or no sensitization (in =226); (ii) highlysensitized (both food and inhalant allergens; n=9); (iii) milk- andegg-dominated (n=50) sensitization or (iv) peanut- andinhalant(s)-dominated (n=13) sensitization. Because of the sample size,latent classes ii-iv were collapsed and considered to be “predominatelymultisensitized” (PM atopy; n=72); remaining subjects represented the“low or no sensitization” group. The conventional definition of atopy(at least one positive test (sIgE≥0.35 IU ml⁻¹) to any of the tenallergens) was also used for comparative purposes. Children were definedas having asthma according to parental-reported doctor diagnosis ofasthma at the 4-year interview.

Bacterial- and fungal-community profiling, PICRUSt and metabolomicanalyses. DNA extraction. Stool samples from 308 infants were extractedby using a modified cetyltrimethylammonium bromide (CTAB)-buffer-basedprotocol¹³. Briefly, 0.5 ml of modified CTAB extraction buffer wereadded to 25 mg of stool in a 2-ml Lysing Matrix E tube (MP Biomedicals,Santa Ana, Calif.) and then incubated (65° C., 15 min). Samples werebead-beaten (5.5 m s⁻¹, 30 s) in a Fastprep-24 (MP Biomedicals, SantaAna, Calif.), which was followed by the addition of 0.5 ml ofphenol:chloroform:isoamyl alcohol (25:24:1). After centrifugation(14,000 rpm, 5 min), the supernatant was added to a heavy phase-lock geltube (5 Prime, Gaithersburg, Md.), and chloroform (v:v) was added.Samples were centrifuged (14,000 rpm, 5 min), and the resultingsupernatants were added to fresh tubes, which was followed by theaddition of 1 μl of linear acrylamide before PEG-NaCl (2v:v). Sampleswere incubated (21° C., 2 h), washed with 70% EtOH and resuspended in 10mM Tris-Cl, pH 8.5.

Sequencing preparation. The V4 region of the 16S rRNA gene wasamplified, as designed by Caporaso et al.¹⁴. PCR reactions wereperformed in 25-μl reactions using 0.025 U Takara Hot Start ExTaq(Takara Mirus Bio Inc, Madison, Wis.), 1× Takara buffer with MgCl₂, 0.4pmol/μl of F515 and R806 primers, 0.56 mg/ml of bovine serum albumin(BSA; Roche Applied Science, Indianapolis, Ind.), 200 μM of dNTPs and 10ng of gDNA. Reactions were performed in triplicate with the following:initial denaturation (98° C., 2 min), 30 cycles of 98° C. (20 s),annealing at 50° C. (30 s), extension at 72° C. (45 s) and finalextension at 72° C. (10 min). Amplicons were pooled and verified using a2% TBE agarose e-gel (Life Technologies, Grand Island, N.Y.), beforeundergoing purification using AMPure SPRI beads (Beckman Coulter, Brea,Calif.), being quality checked with the Bioanalyzer DNA 1000 Kit(Agilent, Santa Clara, Calif.) and being quantified using the Qubit 2.0Fluorometer and the dsDNA HS Assay Kit (Life Technologies, Grand Island,N.Y.). Samples were pooled and sequenced on the Illumina MiSeq platform,as previously described¹⁵.

The internal transcribed spacer region 2 (ITS2) of the rRNA gene wasamplified using the primer pair flTS7 (5′-GTGARTCATCGAATCTTTG-3′) (SEQID NO:7) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (SEQ ID NO:8 Primers weredesigned for the Illumina MiSeq platform, as described above. PCRreactions were performed in triplicate in a 25-μl reaction with 1×Takara buffer (Takara Mirus Bio), 200 nM of each primer, 200 μM dNTPs,2.75 mM of MgCl₂, 0.56 mg ml⁻¹ of BSA (Roche Applied Science,Indianapolis, Ind.), 0.025 U Takara Hot Start ExTaq and 50 ng of gDNA.Reactions were conducted under the following conditions: initialdenaturation (94° C., 5 min), 30 cycles of 94° C. (30 s), annealing at54° C. (30 s), extension at 72° C. (30 s) and a final extension at 72°C. (7 min). PCR verification and purification were performed asdescribed above. Samples were quantified using KAPA SYBR (KAPABiosystems, Wilmington, Mass.) qPCR, following the manufacturer'sprotocol. Samples were pooled in equal moles (50 ng), and prepped anddenatured libraries with PhiX spike-in control, as described above, wereloaded onto the Illumina MiSeq cartridge.

Sequencing-data processing and quality control. For bacterial sequences,paired-end sequences were assembled using FLASH¹⁶ v.1.2.7 andde-multiplexed by barcode, and low-quality reads (Q score, <30) werediscarded in QIIME¹⁷ 1.8. If three consecutive bases were <Q30, then theread was truncated and the resulting read retained in the data set onlyif it was at least 75% of the original length. Sequences were checkedfor chimeras using UCHIME¹⁸ and filtered from the data set beforeoperational taxonomic unit (OTU) picking at 97% sequence identificationusing UCLUST¹⁹ against the Greengenes database²⁰ version 13_5. Inembodiments, closely related microorganisms are grouped together basedon sequence similarity thresholds (e.g., 97%). OTUs represent a userdefined cut off for 16S rRNA sequence identity e.g. 97% identity; allsequences that share at least 97% sequence identity across the sequencedregion of the gene form a single OTU. Sequencing reads that failed tocluster with a reference sequence were clustered de novo. Sequences werealigned using PyNAST²¹, and taxonomy was assigned using the RDPclassifier and Greengenes reference database version²⁰ 13_5. To de-noisethe OTU table, taxa with fewer than five total sequences across allsamples were removed. A bacterial phylogenetic tree was built usingFastTree²² 2.1.3.

Fungal sequences were quality trimmed (Q score, <25) and adaptorsequences removed using cutadapt²³, after which paired-end reads wereassembled with FLASH¹⁶. Sequences were demultiplexed by barcode andtruncated to 150 bp before undergoing clustering using USEARCH vers. 7pipeline, specifically the UPARSE²⁴ function, and being chimera-checkedusing UCHIME. Taxonomy was assigned using UNITE²⁵ vers. 6.

To normalize variation in read depth across samples, data were rarefiedto the minimum read depth of 202,367 sequences per sample for bacteria(n=298) and 30,590 for fungi (n=188). To ensure that a trulyrepresentative community was used for analysis for each sample, sequencesubsampling at these defined depths was rarefied 100 times. Therepresentative community composition for each sample was defined as thatwhich exhibited the minimum average Euclidean distance to all other OTUvectors generated from all subsamplings for that particular sample.Investigators at UCSF were blinded to sample identity until microbiotadata sets underwent the aforementioned processing and were ready forstatistical analyses.

Phylogenetic Reconstruction of Unobserved States (PICRUSt).

PICRUSt⁸ was used to predict the pathways of those taxa significantlyenriched in each NGM state, according to zero-inflated negative binomialregression and corrected for multiple testing using theBenjamini-Hochberg false-discovery rate²⁶(q<0.05). These taxa were usedto generate a new OTU table normalized in PICRUSt, and discriminatorypathways were illustrated in a heat map constructed in R.

Metabolomic Profiling.

Stool samples (200 mg) from each of the three microbiota states, eightNGM3 subjects, and ten from each of NGM1 and NGM2 groups were providedto Metabolon (Durham, N.C.) for ultrahigh performance liquidchromatography-tandem mass spectrometry (UPLC-MS/MS) and gaschromatography-mass spectrometry (GC-MS) using their standard protocol(on the World Wide Web.metabolon.com/). These samples were chosenbecause they exhibited the highest posterior probability of belonging toa given NGM group and possessed sufficient sample volume for UPLC-MS/MSanalysis. Compounds were compared to Metabolon's in-house library ofpurified standards, which includes more than 3,300 commerciallyavailable compounds.

Ex vivo dendritic cell challenge and T cell co-culture. Fecal samplesfrom five of ten NGM1 and seven of eight NGM3 neonates that hadundergone metabolic profiling were used (biological replicates).Excluded samples from these groups had insufficient volume for analyses.Fecal samples were homogenized 1 g ml⁻¹ (w:v) in pre-warmedphosphate-buffered saline (PBS) containing 20% FBS (FBS). Samples werevortexed, incubated (37° C., 10 min) and centrifuged (14,000 rpm, 30min). Supernatant was filter-sterilized through a 0.2-μm filter beforebeing used in the dendritic cell (DC) T cell assay described below. PBSwas used as the negative control. Treatment conditions used for theDiHOME experiment included: 75 μM, 130 μM and 200 μM 12,13 DiHOME(Cayman Chemical, Ann Arbor, Mich.) solubilized in 0.4%, 0.15% and 0.05%DMSO, respectively. DiHOME solutions were added to R10 media (RoswellPark Memorial Institute media 1640 with 10% heat-inactivated FBS(antigen activator) and 2 mM 1-glutamine and 100 U ml⁻¹penicillin-streptomycin added; Life Technologies) and exposed to DCswithin 1 h of preparation. Controls included PBS and DMSO (delivered inR10 media) at corresponding percentages used to dissolve the differentconcentrations of DiHOME. Treatment group size was determined on thebasis of preliminary assays that demonstrated the effect size for thesuppression of CD4⁺CD25⁺FOXP3⁺ using 130 μM of 12,13 DiHOME wasapproximately seven, indicating that at least two samples per group wererequired to achieve a power of >0.80.

Peripheral blood mononuclear cells (PBMCs) were purified from plasmaobtained from healthy, de-identified human donors (Blood Centers of thePacific, San Francisco, Calif.) through the cell-sourcing program thatensures donor confidentiality. Donors signed an agreement acknowledgingthat their blood may be used for research. PBMCs were isolated usingFicoll-Hypaque gradient centrifugation, washed twice with R10 media andincubated for 18 h. Dendritic cells (DCs) were isolated from PBMCs usingthe EasySep Human Pan-DC Pre-Enrichment Kit (STEMCELL Technologies,Vancouver, BC). DCs (0.5×10⁶ cells ml⁻¹) from two donors (biologicalreplication) were treated in triplicate (treatment replicate) witheither cell-free fecal water (0.22 μM filtered) or varyingconcentrations of DiHOME, and cultured in R10 media supplemented with 10ng ml⁻¹ GM-CSF and 20 ng ml⁻¹ IL-4 at 37° C.²⁷ for 2 d, for thefecal-water assay, or 5 d, for the DiHOME experiment. For the DiHOMEexperiment, freshly prepared media containing DiHOME or controlexposures was replaced every 48 h. For the fecal-water experiment, theassay was repeated twice on one donor (technical replicates) and once ondonor B owing to insufficient numbers of cells recovered from the latterdonor. Treatment replicates were also considered biological replicatesbecause the human donor cells are not clonal.

Twenty-four hours before co-culture with CD4⁺ T cells, DC maturation wasstimulated by using DC growth mediators (10 ng ml⁻¹ tumor nuclearfactor-α [(TNF-α), 10 ng ml⁻¹ IL-1b, 10 ng ml⁻¹ IL-6 and 1 mMprostaglandin E2 (PGE2)]. In preparation for co-culture, DCs were washedin fresh R10 media, counted via flow cytometry and plated in TexMACsMedium (Miltenyi Biotec, San Diego, Calif.) at 0.5×10⁶ live CD45⁺ cellsper well.

Autologous T lymphocytes were purified from the PBMCs using a naïve CD4⁺T cell isolation kit (Miltenyi Biotec). After purification, naïveautologous CD4⁺ T cells were suspended in the TexMACS Medium (MiltenyiBiotec) and added to the treated DCs at a ratio of 10:1 in the presenceof soluble anti-CD28 and anti-CD49d (1 mg ml⁻¹). T and DC cells wereco-cultured for 5 d at 37° C. and replenished with fresh TexMACS mediaevery 48 h. To assess cytokine production, the co-cultures were mixedwith Phorbol Myristate Acetate-Ionomycin (SIGSa, St. Louis, Mo.) andGolgiPlug (Gplug; BD Biosciences, San Jose, Calif.) for 16 h before flowcytometry. Cell-free media from the co-cultures was collected at 48 hand 5 d, before PMA-Gplug addition, to assess cytokine secretion.Cytokine secretion was evaluated by cytometric bead array, following themanufacturer's protocol (BD Biosciences).

For flow cytometry, single-cell suspensions were stained using a panelof antibodies, including anti-CD3 (SP34-2, 1:100), anti-CD4 (L200,1:100), anti-CD25 (M-A251, 1:25), anti-IFN-γ (B27, 1:200; BDBiosciences); anti-CD8a (RPA-T8, 1:100; BioLegend, San Diego, Calif.);anti-IL4 (7A3-3, 1:20; Miltenyi Biotec); anti-IL-17A (64DEC17, 1:20) andanti-FOXP3 (PCH101, 1:20; Affymetrix eBioscience, Santa Clara, Calif.).Validation for each primary antibody is provided on the manufacturers'websites. Dead cells were stained positive with LIVE-DEAD Aqua Dead CellStain (Life Technologies). Permeabilization buffer (AffymetrixeBioscience) was used to permeabilize cells before staining for theintracellular markers IFN-γ, IL-4, IL-17A and FoxP3. For flow analysis,live T cells were gated as CD3⁺CD4⁺ cells, wells containing <50% livecells were excluded from analyses. Among CD4⁺ T cell subpopulations, Thelper 1 (Th1) were IFN-γ, T helper 2 (Th2) were IL-4⁺; T helper 17(Th17) were IL-17A⁺, and T regulatory (T reg) cells were both CD25^(hi)and FOXP3^(hi). Stained cells were assayed via a flow cytometer on a BDLSR II (BD Biosciences).

Statistical analysis. Shannon's diversity index was calculated usingQIIME. Pearson's correlation was used to test for a relationship betweenbacterial and fungal Shannon's diversity. Distance matrices (unweightedUniFrac²⁸ and Bray-Curtis) were calculated in QIIME to assesscompositional dissimilarity between samples, and visualized using PCoAplots constructed in Emperor²⁹. Permutational multivariate analysis ofvariance (PERMANOVA) was performed using Adonis in the R environment todetermine factors that significantly (P<0.05) explained variation inmicrobiota β-diversity.

To identify clusters of subjects on the basis of bacterial-taxonomy, DMMmodels were used, which implement an unsupervised Bayesian approach thatis based on a Dirichlet prior⁷. The best-fitting DMM model wasdetermined using the Laplace approximation to the negative-log modelevidence, testing up to ten underlying microbiota states. Each samplewas assigned to a particular neonatal gut microbiota (NGM) state on thebasis of the maximum posterior probability of NGM membership.Kruskal-Wallis was used to test whether age differentiated themicrobiota states. Relative risk (RR) ratios and corresponding 95%confidence intervals were calculated using PROC GENMOD in SAS version9.4 (Cary, N.C.). Unadjusted and adjusted RRs were calculated on thebasis of log-binomial regression using maximum likelihood estimation orrobust Poisson regression, when prevalence ratios were near one or whenthe log-binomial model did not converge. Two-tailed Welch's t test wasused to test whether slgE concentrations (log-transformed) weresignificantly different between the three NGM states.

To determine which OTUs differed in relative abundance between NGMgroups, the zero-inflated negative binomial regression (pscl package)was used as a primary modeling strategy, appropriate for sequence-countdata. In cases in which OTU distributions were not zero-inflated and themodel failed to converge, the standard negative binomial was used as asecondary modeling strategy. These were corrected for multiple testingusing the minimum positive false-discovery rate (q<0.05 for bacteria;q<0.20 for fungi)²⁶. Results were natural-log transformed forillustration on phylogenetic trees using iTOL³⁰ v.3.0. When examiningthe association between early-life factors and NGMs, P values werecalculated on the basis of covariate distribution by ANOVA (numerical,normally distributed), Kruskal-Wallis (numerical, skewed), chi-square(categorical) or Fisher's exact (sparse categorical).Log-binomial-regression model was used to test for confounding factorswhen assessing the RR of individuals with different microbiota statesdeveloping atopy or asthma (PROC GENMOD in SAS version 9.4). Fisher'sexact two-tailed test was conducted to test whether breastfeeding waspracticed significantly (P<0.05) more often in any particular NGM.

Metabolites exhibiting significantly (P<0.05) different concentrations(log-transformed) between lower-risk NGM states and NGM3 were identifiedusing two-tailed Welch's t test. Shared and distinct super- andsub-pathway products among NGMs were illustrated using Cytoscape, vers.3.2.1 (ref. 31). Co-occurrence networks of metabolites were constructedusing weighted correlation network analysis (WGCNA) with the R packageWGCNA to find modules of highly interconnected, mutually exclusivemetabolites. Pearson correlations were used to determine intermetaboliterelationships, wherein modules are composed of positively correlatedmetabolites. To avoid spurious modules, the minimum module size was setto five. Module ‘eigenmetabolites’ (referred to as eigengenes) weredefined as the first principal component of a given module andconsidered as a representative measure of the joint metabolic profile ofthat module. Each eigenmetabolite was used to test (ANOVA) theassociation between its respective module and NGM, module membership wasused to determine the interconnectedness of each metabolite to itsassigned module and to identify ‘hub’ metabolites: this was defined asthe correlation between each metabolite and the eigenmetabolite (strongpositive values indicate high interconnectedness).

Procrustes was used to test for concurrence between communitiesdescribed by 16S phylogeny, PICRUSt and metabolomics data sets.

To test for T cell and cytokine differences, a linear mixed-effectsmodel (LME) was used (R package ImerTest) and adjusted for donors.Except where indicated, all analyses were conducted in the R statisticalprogramming language.

AOP: Differences in the composition of the gut microbiota of infantsassociate with relative risk of atopy in childhood, and metaboliteslinked with these distinct microbial states alter T cell differentiationin vitro.

Issue: Differences in the composition of the gut microbiota of infantsassociate with relative risk of atopy in childhood, and metaboliteslinked with these distinct microbial states alter T cell differentiationin vitro.

Gut microbiota-state validation in an independent cohort. To assess thevalidity of our DMM modeling, the published 16S rRNA data of Arrieta etal.¹ was used (n=319 independent fecal samples collected atapproximately 3-12 months of age in the Canadian Healthy InfantLongitudinal Development (CHILD) Study). The specific age of eachparticipant was unavailable and the youngest participants in this cohortwere 3 months of age, substantially older than neonates in the WHEALScohort. Hence the dataset could not be segregated into samples thatwere > or <6 months of age, as had been performed for our Wayne CountyHealth, Environment, Allergy and Asthma Longitudinal Study (WHEALS)cohort. This limited our capacity to identify neonatal microbiota statesassociated with subsequent childhood atopy and asthma outcomes.Nonetheless, we used the cohort to determine whether any of themicrobiota states identified in our study were replicated in the CHILDcohort. Because of the age range of the CHILD cohort, we applied bothour NGM and IGM model parameters to the entire data set. A better modelfit (i.e., smaller laplace approximation to the negative log modelevidence) was obtained when the CHILD data was fit to the NGM modelcompared with the IGM model (model fit: 32,502 versus 174,610,respectively) and a two-group solution represented the best fit for theCHILD data. Group 1 (G1) included 221 (69%) participants and group 2(G2) 98 (31%). The posterior probabilities were on average higher for G1compared to G2 (0.98 vs. 0.95, respectively). Consistent with ourfindings, CHILD participants assigned to G1 were typically defined byhigh Bifidobacteriaceae relative abundance (average relative abundance(aRA): 75%). G2 participants were characterized by Lachnospiraceae (aRA:39%), Clostridiaceae (aRA: 290%), and Ruminococcaceae (aRA: 12⁰%), morereflective of the IGM2 cluster identified in our cohort.

Code Availability.

The following script may be used to calculate a representative multiplyrarefied OTU table from an unrarefied OTU table, an alterative to singlyrarefied tables. This approach stabilizes the effect of random samplingand results in an OTU table that is more representative of communitycomposition. Multiple single-rarefied OTU tables are calculated for eachsample, and the distance between the subject-specific rarefied vectorscalculated. The rarefied vector that is the minimum average (or median)distance from itself to all other rarefied vectors is considered themost representative for that subject and used to represent communitycomposition for that sample in the resulting multiply-rarified OTUtable.

library(vegan) library(GUNifFrac)

## Parameters

# specify the raw OTU count table, with samples=rows, taxa=columns #rawtab=otu_tab_t

# specify the depth you would like to rarefy your tables to the defaultis to just use the minimum sequencing # depthraredepth=min(rowSums(rawtab))

# specify the number of rarefied tables you would like to generate tocalculate your representative rarefied # table from ntables=100

# specify the distance measure to use to calculate distance betweenrarefied data sets, for each subject

# can be any of the methods available in the vegdist function of vegandistmethod=“euclidean”

# specify the method to summarize across distances if mean distance,then summarymeasure=mean

# if median distance, then summarymeasure=median

# summarymeasure=mean

# specify the seed start for the rarefied tables

# for each subsequent table, 1 will be added that the previous seed

# for reproducibility, always save your seedstart value (or just use thedefault for simplicity).

# seedstart=500

# specify if you want progress updates to be printed # verbose

=TRUE ## returns a representative rarefied OTU table of

class matrix.## functions

reprare<-function(rawtab=otu_tab_t, raredepth=min(rowSums(otu_tab_t)),ntables=100, distmethod=euclidean”,

summarymeasure=mean, seedstart=500, verbose=TRUE) {

raretabs=list( )

for (z in 1:ntables) {

if (verbose==TRUE) {

print(paste(“calculating rarefied table number”, z, sep=“ ”))

}

set.seed(seedstart+z)

raretabs[[z]]=Rarefy(rawtab, depth=raredepth)[[1]]

}

raretabsa=array(unlist(raretabs), dim=c(nrow(raretabs[[z]]),ncol(rawtab), ntables))

final_tab=c( )

for (y in 1:nrow(raretabs[[z]])) {

if (verbose==TRUE) {

print(paste(“determining rep rarefied vector for subject number”, y,sep=“ ”))

}

distmat=as.matrix(vegdist(t(raretabsa[y,]), method=distmethod)) #distance across reps for subject y

distsummary=apply(distmat, 2, summarymeasure)

whichbestrep=which(distsummary==min(distsummary))[1] # the best rep isthe one with the minimum average/median distance to all other reps. (incase of ties, just select the first)

bestrep=raretabsa[y, whichbestrep] # select that rep only for subject y

final_tab=rbind(final_tab, bestrep) # build that rep for subject y intofinal table

}

rownames(final_tab)=rownames(raretabs[[z]])

colnames(final_tab)=colnames(rawtab)

return(final_tab)

}

###### example runs of the function: ######

### dummy data set for example ###

ntaxa=200

nsubj=50

set.seed(444)

dummyOTU<-matrix(sample(0:500, ntaxa*nsubj, prob=c(0, 7, 0, 1, 0.1,rep(0.1/498, 498)),

replace=TRUE), ncol=ntaxa)

colnames(dummyOTU)=paste(“OTU”, 1:ntaxa, sep=“ ”)

rownames(dummyOTU)=paste(“subj”, 1:nsubj, sep=“ ”)

sort(rowSums(dummyOTU)) # sequencing depth is uneven

# specify the minimum depth

repraretable=reprare(rawtab=dummyOTU, raredepth=min(rowSums(dummyOTU)),

ntables=100, distmethod=“euclidean”,

summarymeasure=mean, seedstart=500, verbose=TRUE)

dim(repraretable)

sort(rowSums(repraretable)) # sequencing depth is now even

# specify a depth other than the minimum

repraretable=reprare(rawtab=dummyOTU, raredepth=3380, ntables=100,distmethod=“euclidean”,

summarymeasure=mean, seedstart=500, verbose=TRUE)

dim(repraretable) # subjects with less than the minimum are no longer inthe table

sort(rowSums(repraretable)) # sequencing depth is now even

Example 5: Disease Severity and Immune Activity Relate to DistinctInterkingdom Gut Microbiome States in Ethnically Distinct UlcerativeColitis Patients

Significant gut microbiota heterogeneity exists among ulcerative colitis(UC) patients, though the clinical implications of this variance areunknown. We hypothesized that ethnically distinct UC patients exhibitdiscrete gut microbiotas with unique metabolic programming thatdifferentially influence immune activity and clinical status. Usingparallel 16S rRNA and internal transcribed spacer 2 sequencing of fecalsamples (UC, 30; healthy, 13), we corroborated previous observations ofUC-associated bacterial diversity depletion and demonstrated significantSaccharomycetales expansion as characteristic of UC gut dysbiosis.Furthermore, we identified four distinct microbial community states(MCSs) within our cohort, confirmed their existence in an independent UCcohort, and demonstrated their coassociation with both patient ethnicityand disease severity. Each MCS was uniquely enriched for specific aminoacid, carbohydrate, and lipid metabolism pathways and exhibitedsignificant luminal enrichment of the metabolic products of thesepathways. Using a novel ex vivo human dendritic cell and T-cellcoculture assay, we showed that exposure to fecal water from UC patientscaused significant Th2 skewing in CD4⁺ T-cell populations compared tothat of healthy participants. In addition, fecal water from patients inwhom their MCS was associated with the highest level of disease severityinduced the most dramatic Th2 skewing. In embodiments identification ofhighly resolved UC subsets based on defined microbial gradients ordiscrete microbial features are exploited for effective therapies.

Despite years of research, the etiology of UC remains enigmatic.Diagnosis is difficult and the patient population heterogeneous, whichrepresents a significant barrier to the development of more effective,tailored therapy. In this study, we demonstrate the clinical utility ofthe gut microbiome in stratifying UC patients by identifying theexistence of four distinct interkingdom pathogenic microbiotas withinthe UC patient population that are compositionally and metabolicallydistinct, co-vary with clinical markers of disease severity, and drivediscrete CD4⁺ T-cell expansions ex vivo. These findings offer newinsight into the potential value of the gut microbiome as a tool forsubdividing UC patients, opening avenues to the development of morepersonalized treatment plans and targeted therapies.

Though murine and human studies support the involvement of the gutmicrobiota in the development and pathogenesis of ulcerative colitis(UC; a common form of inflammatory bowel disease [IBD]), a singlecausative microbial agent has not been identified and depletion ofbacterial diversity remains the primary constant feature of UC gutmicrobiome dysbiosis (1). Increasingly, disease endotypes have beendescribed among patients within clinically defined chronic inflammatorydiseases (2), suggesting that, in the context of immune dysfunction,distinct pathogenic processes may converge upon a common clinicaldisorder. Since UC pathogenesis is related to gut microbiomecomposition, we rationalized that factors that dictate the compositionand function of these communities may lead to the development ofdistinct gut microbiome states that function as discrete pathogenicunits to deterministically influence immune activation status anddisease severity.

Host genetics, diet, and environmental exposures, three factorsencompassed by ethnicity, influence both the gut microbiome and UCpathology (3). Indeed, healthy subjects in the United States, Venezuela,and Malawi exhibit a significant relationship between ethnicity and boththe composition and function of the fecal microbiota, with dietrepresenting strong selective pressure on the gut microbial assemblage(4). Independently, Frank et al. demonstrated that in a U.S. cohort, IBDrisk alleles ATG16L1 and NOD2 (associated with autophagy and the hostresponse to microbes, respectively) are significantly associated withgut microbiome β diversity (5). However, a meta-analysis of genome-wideassociation studies indicated that such UC risk alleles characteristicof Caucasian populations do not confer a heightened risk on ethnicallydistinct north Indian subjects (6). In embodiments, distinct pathogenicmicrobiotas exist within UC patients that covary with both patientethnicity and disease severity. In embodiments, these distinctpathogenic microbiotas exhibit a predictable program of luminalmetabolism that induces significantly different degrees of Th2activation.

Results. Interkingdom gut microbiota perturbations are characteristic ofUC patients. Our study population consisted of a cohort of 43 subjects(30 UC patients and 13 healthy subjects) of self-reported European orSouth Asian (SA) ethnicity. Several studies have examined bacterialcommunity composition in fecal samples from UC patients; however, todate, none have examined the mycobiome of adult UC patients. Usingparallel, high-resolution bacterial (16S rRNA) and fungal (internaltranscribed spacer 2 [ITS2]) biomarker gene profiles, we confirmed thatour ethnically restricted UC population exhibited bacterial microbiotadysbiosis consistent with that previously described (1). Compared tohealthy subjects, UC patients had significantly reduced a diversity(P=0.010; FIG. 31A) and were compositionally distinct (permutationalmultivariate analysis of variance [PERMANOVA]: weighted UniFrac,R²=0.058, P=0.023) (FIG. 31B). Neither fungal α- or β-diversity differedbetween healthy and UC patients (P=0.523; see FIG. 34A) (PERMANOVA:Bray-Curtis, R²=0.038, P=0.129; see FIG. 34B), indicating that whileprofound bacterial depletion is characteristic of the UC gut microbiota,more subtle changes in fungal taxonomy characterize these patients.

A total of 165 bacterial taxa were significantly differentially enrichedin healthy participants and UC patients. Consistent with previousreports, specific Bacteroides and Prevotella species and a number ofunclassified members of the families Lachnospiraceae and Ruminococcaceaewere among the bacterial taxa most significantly depleted in UC gutmicrobiotas (8, 9). UC patients also exhibited enrichment of members ofthe Streptococcus, Bifidobacterium, and Enterococcus genera, which wasvalidated by independent phylogenetic microarray profiling of these samesamples and confirms previous reports (8, 9). Only a small number offungal taxa (n=13) exhibited differential relative abundance. UCpatients were depleted of Alternaria alternata, Aspergillus flavus,Aspergillus cibarius, and Candida sojae while being significantlyenriched in Candida albicans and Debaryomyces species. Collectively,these data indicate that the UC-associated gut microbiota ischaracterized by an interkingdom dysbiosis, highlighted by significantexpansion of putatively pathogenic bacterial and fungal species, in thecontext of depleted bacterial diversity.

UC fecal microbiotas segregate by ethnicity, dominant microbialfeatures, and disease characteristics. We next addressed our hypothesisthat ethnicity is associated with distinct interkingdom fecal microbiotain UC patients. Healthy EU and SA participants exhibited no significantdifference in bacterial or fungal a diversity (see FIG. 34C and FIG.34D). However, SA-UC patients consistently exhibited less bacterialdiversity than either healthy ethnically matched controls or EU UCpatients (see FIG. 34C). They also were significantly depleted of fungaldiversity compared to the EU UC group (see FIG. 34D), indicating moresevere interkingdom microbiome depletion in these patients, though nodifference in clinical disease severity between EU and SA-UC patientswas observed (see FIG. 34E). Ethnicity was also significantly associatedwith bacterial, but not fungal, β diversity when all of the participantswere considered (see FIG. 34F and FIG. 34G). Because health status wassignificantly associated with gut microbial composition (FIG. 31B), itrepresented a potential confounding factor. We therefore repeatedPERMANOVA with only UC patients and showed that, while fungal communitycomposition does not exhibit a significant relationship with patientethnicity (PERMANOVA: Bray-Curtis, R²=0.061, P=0.107), bacterial βdiversity does (PERMANOVA: weighted UniFrac, R²=0.075, P=0.039; FIG.31C), an observation validated by PhyloChip data (see FIG. 34H). Thus,these data indicate that, despite chronic colonic inflammatory disease,ethnicity remains associated with compositionally distinct bacterialcommunities in the UC gut, though it explains only a small proportion(7.5%) of the observed variation in β diversity across these patients.

Recent pediatric Crohn's disease studies have demonstrated that patientscluster into subgroups based on patterns of microbial coassociation (10,11). We next asked whether such patterns exist in our adult UC cohortand relate to patient ethnicity and/or clinical correlates of diseaseseverity. Using hierarchical cluster analysis and multiscale bootstrapresampling, we identified four subgroups of UC patients based on fecalbacterial community composition and termed these microbial communitystate 1 (MCS1) to MCS4. These distinct patient subgroups were confirmedby PERMANOVA with both 16S rRNA sequence and PhyloChip data (see FIG.35A and FIG. 35B). MCS distribution differed significantly acrossethnicities, with EU UC populations primarily composed of MCS1 and MCS2while SA UC patients exhibited a relatively equal distribution of allfour MCSs (Fisher exact test, P=0.042).

The clinical relevance of grouping patients on the basis of MCSs wasassessed by using an intergroup comparison of clinical disease severity(simple clinical colitis activity [SCCA] index) (12), extracolonicmanifestations (arthritis, pyoderma gangreno-sum, erythema nodosum, anduveitis), the number of first- and second-degree relatives diagnosedwith IBD, and duration (years since UC diagnosis). MCS1 patientsexhibited more severe disease with higher median SCCA scores, asignificant increase in the number extracolonic manifestations, agreater number of first- and second-degree relatives diagnosed with IBD,and longer disease duration (FIG. 32). Though the number of patients inour study is small, these data provide the first indication thatdistinct pathogenic UC gut microbiotas exist and are associated withclinical features of disease severity.

UC MCSs exhibit distinct taxonomic enrichments, metag-enomic capacity,and metabolic productivity. The distribution of microbial taxa acrossthe four UC MCSs was assessed to identify specific bacterial and fungalenrichments characteristic of each. Each MCS typically exhibited adistinct dominant bacterial family (MCS1, Bacteroidaceae; MCS2,Lachnospiraceae, Ruminococcaceae; MCS3, Prevotellaceae; MCS4,Bifidobacteriaceae). These MCS-specific bacterial enrichments extendedbeyond the dominant family and were further emphasized when the highestdisease severity group (MCS1) was compared to each of the other threegroups (MCS2, -3, or -4). Specifically, a majority of the bacterial taxaenriched in MCS1 were members of the Bacteroides genus, while the othersubgroups were enriched for Blautia, Ruminococcus (MCS2), Prevotella(MCS3), or Bifidobacterium (MCS4, generalized linear models, P<0.05)species. Using the dominant bacterial family as a classifier, wevalidated the existence of MCS1 and −2 (the two major MCSs in EU UCpatients) in two publicly available UC microbiota data sets obtainedfrom patients primarily of European descent (9, 11), indicating thatthese MCSs are not exclusive to our study but exist in UC patientpopulations nationwide. Mycologically, C. albicans and Debaryomycesspecies were most highly enriched in MCS1 patients compared to each ofthe other three MCSs (generalized linear models, P<0.05), indicatingthat interkingdom gut microbiome expansion of Bacteroides species, C.albicans, and Debaryomyces species is associated with more severe UCdisease.

To identify microbiota-derived pathways and products characteristic ofeach MCS that may modulate the host immune response and contribute toclinical disease severity, we performed in silico metagenomicpredictions in parallel with broad-spectrum gas and liquidchromatography mass spectrometry of fecal samples. Phylogeneticinvestigation of communities by reconstruction of unobserved states(PICRUSt; picrust.github.io/picrust/) (13) was used to predict bacterialfunctional capacity. Presently, this algorithm cannot be used to predictfungal community function. Predicted metabolic capacity variedsignificantly by MCS (PERMANOVA: Bray-Curtis, R²=0.384, P=0.002). Atotal of 144 bacterial KEGG pathways discriminated MCS1 to −4, includingthose involved in amino acid and lipid biosynthesis and metabolism(Kruskal-Wallis test, q<0.0006). Specifically, differential enrichmentof glycerolipid, fatty acid, inositol, and multiple amino acidmetabolism pathways, including phenylalanine, tyrosine, tryptophan,glutamate, and glutamine, differentiated these groups. We also generatedfunctional predictions for MCS1 and -2 stool samples from the studies ofMorgan et al. and Gevers et al. (9, 11). A total of 121 KEGG pathwayswere differentially enriched between MCS1 and MCS2 in our study; ofthese, 74 (61.2%) also discriminated MCS1 from MCS2 in both the datasets of Gevers et al. and Morgan et al., indicating a high degree ofconserved microbial function associated with MCS1 and −2 across multipleindependent studies.

We hypothesized that the predicted functional differences across MCSswould be manifested as distinct programs of luminal metabolism,particularly since the majority of the pathways that differentiatedthese communities were involved in amino acid and lipid metabolism.Indeed, each MCS exhibited significantly distinct metabolic programs(PERMANOVA: Canberra, R²=0.209, p=0.004) that were significantly relatedto both the fecal microbiota present (Mantel test, r=0.38, P<0.0001) andits predicted metagenome (Mantel test, r=0.21, P<0.008). We wereparticularly interested in those luminal metabolites that discriminatedthe more severe MCS1 from each of the remaining MCSs. Of the 805metabolites detected across all of the samples, 207 exhibitedsignificant inter-MCS differences in relative concentration (Welch's ttest, P<0.05). Compared to MCS groups with lower disease severity, MCS1,as our in silico predictions suggested, was significantly enriched forophthalmate (a biomarker of increased oxidative stress and depletedglutathione) (14), oxidative-stress-inducing putrescine (15),proinflammatory p-cresol sulfate (16), 9-hydroxyoctadecadienoic acid and(9-HODE) and 13-HODE (a proinflammatory, leukocyte-recruitingmonohydroxy fatty acid) (17, 18), and 9,10-dihydroxyoctadecanoic acid(9,10-DiHOME; a neutrophil-recruiting, cytotoxic dihydroxy fatty acid)(19), as well as bioactive lysolipids involved in leukocyte activation(FIG. 33) (18, 20). In contrast, lower disease severity MCSs (MCS2, -3,and -4) were enriched for a range of potentially protective dipeptides(including anti-inflammatory alanyl-glutamine) (21, 22), γ-glutamyldipeptides indicative of improved oxidative stress coping mechanisms(23), and antioxidant immunosuppressive myo-inositol (24, 25). Theseobserved differences in gut luminal metabolic programming between MCSsassociated with high and low UC severities indicate the existence ofputative mechanisms to control inflammation in patients with less severedisease.

T-cell activity in vitro is related to MCS and health status. Recentstudies have demonstrated that specific gut microbiome-derivedmetabolites influence Th2 responses (7) and, independently, thatproinflammatory cytokine production by T-helper cell populations,including Th2 cells, is a characteristic of UC (26). We thereforehypothesized that the luminal milieus associated with distinct MCSsdifferentially influence CD4⁺ T-cell activation in a manner consistentwith disease severity. To assess this, we developed an ex vive assayinvolving coincubation of human dendritic cells (DCs; obtained fromhealthy donors) with filter-sterilized fecal water prepared from studyparticipants' feces. DCs were then cocultured with autologous CD4⁺ Tcells prior to analyses of T-cell phenotypes and cytokine productivity.Compared to healthy participants, UC patients exhibited a significantreduction in the ratio of Th1 to Th2 cells, significantly increasednumbers of both Th1 and Th17 cells, and trends toward increases in bothT-regulatory and Th2 cell populations (linear mixed effects, P<0.05)(FIG. 33A-33E). CD8 T-cell subsets did not differ significantly betweenhealthy participants and UC patients (data not shown). These findingssuggest that luminal microbial products captured in sterile fecal watercontribute to UC by inducing a Th2-skewed expansion of CD4⁺ T-cellpopulations.

Having demonstrated the Th2-skewing effect of UC-associated fecal water,we next asked if this immune response varied on the basis of MCS andassociated differences in symptom severity, focusing specifically on Th1and Th2 populations. With the exception of a minor significant increasein Th1 populations in response to MCS4 fecal water, no significantdifferences in overall Th1 or Th2 cell populations were observed betweenMCS groups and controls (FIG. 33F and FIG. 33G). However, when theTh1-to-Th2 ratio was calculated for each group, the MCS1 groupexclusively exhibited a significantly lower Th1-to-Th2 ratio comparedwith healthy controls (FIG. 33H). Of note, no difference in theTh1-to-Th2 ratio was observed when UC patients were compared on thebasis of ethnicity (EU UC versus SA UC, see FIG. 36), providing evidencethat patient ethnicity alone is not responsible for the altered T-cellactivity observed ex vivo. Furthermore, when considering the two MCSsdemonstrating the greatest difference in disease severity (MCS1 andMCS2), only MCS1 fecal water significantly increased secretion ofTh2-associated cytokines compared with healthy controls (FIG. 33I-33K).These ex vivo data provide evidence that compositionally andmetabolically distinct UC microbiotas are capable of differentiallyinfluencing CD4⁺ T-cell populations in a manner consistent with UCdisease severity.

Discussion Heterogeneity among UC patients is poorly understood andrepresents a significant barrier to more effective therapy. Colitisdevelopment necessitates microbial involvement, and gut micro-biomedysbiosis is characteristic of adult UC patients, but while genetic,therapeutic, and environmental factors are related to UC bacterial βdiversity, they explain a small proportion of the observed variation inthese microbial communities (5, 9). Microbial species engage in inter-and intraspecies interactions that dictate coassociated microbes andtheir physiology (27, 28). For example, C. albicans coaggregates withspecific bacterial species in the oral microbiota, facilitating morerobust, stress-resistant mixed-species biofilms (27). In turn, theproducts of these coassociated bacteria induce a physiological shifttoward unicellular morphology in C. albicans (27). Similarly, because ofmetabolic cross-feeding, Streptococcus gordonii facilitatescoassociation with Fusobacterium nucleatum (28). Hence, we rationalizedthat, under the proinflammatory conditions of the colitic gut, distinctpatterns of pathogen coassociation occur whose composition and functionare relatively conserved across patients and related to immuneactivation and disease severity. Our data support the existence of fourdistinct UC MCSs that differ significantly in their prevalence alongethnic divides. Internal and external validation confirmed the existenceof the predominant microbiota states, indicating that, despite inherentpatient variability, treatment regimens, and geography, conservedpatterns of pathogenic microbiota coassociation exist across UCpopulations within the United States. To improve our understanding ofthe progression and development of these MCSs, it will be important forfuture studies to investigate UC patient factors, be they temporal,clinical, genetic, or environmental, that directly drive the microbiometoward these differential microbial states.

Of the four MCSs identified in our study, MCS1 represented the most illpatient group, implicating the composition and metabolism of MCS1 inenhanced immune activation and increased disease severity. MCS1characteristically exhibited expansion of Bacteroides species, which canproduce enterotoxin previously associated with UC, stimulateinterleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α) secretionin intestinal epithelial cells, and intensify colitis symptoms in amurine model of UC (29-31). MCS1 patients also exhibited the greatestexpansion of C. albicans and Debaryomyces species. Gut microbialexpansion of these fungal species has also been described in adult andpediatric Crohn's disease, as well as pediatric IBD (Crohn's disease andUC patients combined) (10, 32, 33). Together with our study, these dataindicate that expansion of Saccharomycetales fungi in the context ofdepleted bacterial diversity is a consistent feature of IBD in pediatricand adult populations. Whether C. albicans directly influences UCpathology in patients in our study is unclear. However, gastrointestinalcolonization by C. albicans impairs gastrointestinal healing in both UCpatients and a murine model of UC and can induce a Th2 responsefollowing gastrointestinal infection of mice with antimicrobial-depletedgut microbiota diversity (34, 35).

The MCS2 subgroup was enriched for both Blautia and Rumi-nococcusspecies, which together may produce anti-inflammatory short-chain fattyacids (36-38). Prevotella species (enriched in MCS3) are capable ofsuppressing lymphocyte activity, while Bifidobacterium species (enrichedin MCS4) can reduce the production of both IL-8 and TNF-α in intestinalepithelial cells (39, 40). It should be noted that one patient in ourstudy, who demonstrated a dramatic enrichment of Porphyromonadaceae (seeFIG. 35A and FIG. 35B), was not classified as having one of the fourmain MCSs identified here and, though removed from our analysis, mayrepresent an additional, clinically relevant MCS that, given additionalpatient enrollments, future studies may further characterize and drawconclusions from. Though confirmation that the MCSs identified in ourstudy are also present in independent UC microbiome studies indicatesthe relative durability of these microbial states, their long-termstability cannot be assessed in cross-sectional studies. It is likelythat these MCSs represent discrete points along a nonlinear continuum ofpathogenic microbial successional states that relate to diseaseprogression and severity, similar to the microbial gradient identifiedby Gevers et al. in pediatric Crohn's disease (11). Though thesecross-sectional studies are informative, more expansive, longitudinalstudies are necessary to determine the natural history of the gutmicrobiome in UC development and progression.

While interkingdom microbial taxonomic states represent an economicalmeans to stratify patients in large studies, the functional capacity andproductivity of these compositionally discrete pathogenic microbiota areparamount to dictating host immune responses and clinical diseaseseverity. Indeed, in our study, programs of metabolic productivityidiosyncratic to the predicted pathways encoded by bacteria present ineach MCS were identified. In particular, 9-HODE, 13-HODE, 9,10-DiHOME,and lyso-phosphatidylcholines (significantly enriched in MCS1) canincrease leukocyte recruitment and proinflammatory cytokine secretion(17-20). Soluble epoxide hydrolase inhibitors, which prevent 9,10-DiHOMEformation, attenuate UC in both chemical and genetic murine models (41),underscoring a potential role for these oxylipins as contributors tomore severe disease and that treatments inhibiting their production maybe especially efficacious in this specific patient subgroup. In additionto enrichment of leukocyte chemotactic metabolites, MCS1 patients alsohad high fecal concentrations of p-cresol sulfate, a microbe-derivedmetabolite (42), and putrescine, both of which can stimulate a leukocyteoxidative burst (15, 16). Consistent with these observations,ophthalmate was also enriched in MCS1 patients, indicative of greateroxidative stress due to low or depleted levels of reactive oxygenspecies (ROS) quenching glutathione (14). While the metabolome of highdisease severity MCS1 indicated conditions of high oxidative stress,that of UC MCSs associated with lower disease severity (MCS2 to -4)exhibited an increased capacity for ROS quenching due to enhancedγ-glutamyltransferase activity indicated by enrichment of γ-glutamylamino acids (critical for maintaining glutathione levels) and highconcentrations of superoxide scavenging myo-inositol (23, 24). Metabolicsignatures indicative of immunosuppressive activity, such as enrichmentof anti-inflammatory dipeptides (i.e., alanyl-glutamine) andmyo-inositol (both of which decrease the expression of proinflammatorycytokines and reduce leukocyte recruitment in animal models of colitis)(21, 22, 25), were also observed in MCS2 to −4 with lower diseaseseverity. This suggests that the specific metabolic productivityassociated with each MCS may govern host immune activity and resultingdifferences in UC severity.

MCS-associated luminal products, which include host- and/ormicrobe-derived immunomodulatory metabolites, provide a multifacetedmechanism by which a pathogenic gut microbiota may influence hostphysiology and dictate clinical disease severity. Thoughpathogen-associated molecular patterns (PAMPs) have traditionally beenconsidered paramount to driving host immune responses to microbes,emerging data in the field of immuno-metabolism indicate thatmicrobe-derived metabolites are equally effective in dictating immunecell phenotypes. In addition to the established direct immunomodulatoryactivity of microbe-derived metabolites such as short-chain fatty acidsor p-cresol sulfate (16, 38), recent studies have demonstrated that thegut microbiota-associated metabolites taurine, histamine, and sperminecomodulate NLRP6 inflammasome signaling, epithelial IL-18 secretion, anddownstream antimicrobial peptide production (43). Indeed, our datasuggest that specific programs of microbe-derived metabolism incombination with an array of PAMPs presented by pathogenic bacteria andfungi in the distal gut of UC patients serve as effective drivers ofimmune dysfunction related to UC disease severity. Support for thisconcept comes from our demonstration ex vivo that sterile fecal waterfrom the most severely ill MCS1 patients induced the greatest degree ofTh2 skewing in T-cell populations and associated cytokine production, afeature not observed among the other subgroups with less severe disease.While this observation does not directly implicate the microbiome as acausative agent of UC, it does provide evidence of the ability of themicrobiome to perpetuate the inflammation and symptoms associated withUC in a manner specific to microbiota composition. This finding alsoindicates that the Th2 skew traditionally considered characteristic ofUC patients (26) is not a consistent finding across our cohort and may,in fact, be driven by the most severely ill patients in UC cohorts(i.e., MCS1). Whether or not different inflammatory phenotypes presentamong UC patients select for phenotype-maintaining microbes or are theresult of initial, discrete dysbioses remains to be addressed.Regardless, this raises the possibility that distinct immunologicalfeatures not examined in this study characterize patients with lowerdisease activity and distinct gut MCSs. Future larger studies will beimportant in further characterizing the potential immuno-modulatorycontributions of theses MCSs while confirming the observations presentedhere. Hence, therapies tailored to the specific microbial, metabolic,and immune dysfunctions exhibited by UC patient subgroups may prove ahighly efficacious strategy for more effective treatment of thisdisease.

Materials and Methods. Fecal sample collection and nucleic acidisolation. Stool samples were collected from healthy participants andphysician-diagnosed UC patients of either EU or SA ethnicity by using astandardized protocol. Fecal DNA was extracted with a combination ofbead beating and the commercially available QIAamp DNA Stool kit(catalog no. 51504; Qiagen, CA).

Bacterial 16S rRNA profiling. Total DNA extracted from fecal samples wasused as the template for 16S rRNA gene amplification (in triplicate)with barcoded primers targeting the V4 region as previously described(44). Sequencing libraries were created as previously described (44).Full-length 16S amplicons were also generated and hybridized to the G316S rRNA PhyloChip (Affymetrix, CA) as previously described (45).

Fungal ITS2 library preparation. ITS2 sequencing libraries were createdwith triplicate PCR amplicons per sample.

16S and ITS2 library sequencing Purified sequencing libraries wereanalyzed with a Bioanalyzer (Agilent), quantified with the Qubit HSds-DNA Assay kit (Invitrogen), and sequenced with an Illumina MiSeqplatform and MiSeq Control Software v2.2.0 according to themanufacturer's instructions (Illumina). FLASH v1.2.7, QIIME 1.8, andusearch software packages were used for sequence read quality filtering,operational taxonomic unit (OTU) picking, and OTU table generation(46-48).

Predicted community metagenome analyses. PICRUSt(picrust.github.io/picrust/) was used to generate in silico bacterialmetag-enomes by using 16S rRNA data (13).

Metabolome profiling. To profile fecal metabolites, >200 mg of eachfrozen stool sample was shipped overnight on dry ice to Metabolon, Inc.(Durham, N.C.), for broad-spectrum gas and liquid chromatography-massspectrometry.

In vitro DC/T-cell fecal water assay. DCs obtained from anonymoushealthy human donors (Blood Centers of the Pacific) were coincubated for24 h with fecal water prepared from the same fecal samples submitted formetabolite profiling (filter to remove intact cells) prior tostimulation with TNF-α, IL-1f3, IL-6, and prostaglandin E2 and incubatedfor an additional 24 h to induce maturation. DCs were then harvested,washed, and cocultured with autologous T cells at a ratio of 1/10 for 5days, with medium replenishment every 2 days. The T-cell phenotype wasassessed via flow cytometry, and cytokine secretion was assessed byCytometric Bead Array analysis (BD Biosciences). The assay was repeatedin quadruplicate with distinct donors to ensure that observations werenot confounded by the peripheral blood mononuclear cell (PBMC) source.

Statistical analysis. (i) Microbial, metagenomic, and metabolomicanalyses. Statistical analyses were performed with QIIME v1.8.0 and theR statistical environment (47, 49). For PhyloChip data, fluorescenceintensities were log normalized prior to analysis. (ii) Comparison ofclinical measurements of disease severity. Clinical measurements ofdisease severity were compared between UC MCSs by a Kruskal-Wallis test,followed by a pairwise two-tailed Dunn test. (iii) Analysis of T-cellsubsets. A linear mixed-effect model was applied with the lme4 packagein R to identify significant differences in the abundance of inducedT-cell subpopulations based on sample groups (i.e., UC MCSs) whileaccounting for potential variation introduced by the PBMC source (i.e.,donor) (50).

Microarray and nucleotide sequence data accession numbers. Allmicroarray data have been deposited in the Gene Expression Omnibusdatabase (ncbi.nlm.nih.gov/geo) under accession no. GSE78724. All of thesequence data related to this study are available in the Sequence ReadArchive database (ncbi.nlm.nih.gov/sra) under accession no. SRP071201.

Fecal sample collection. Study participants were provided detailedinstructions and necessary materials for fecal sample collection.Standardized fecal samples (first stool of the morning) were collect athome by defecating onto a sterile stool collection device (Cat. No.Protocult #120; Ability Building Center, MN) placed over a toilet seatand using a sterile collection cup with an attached sterile scoop (Cat.No. 80.734.311; Sarstedt, Germany). Following collection, fecal sampleswere placed in a pre-paid overnight mailer with a frozen ice pack (Cat.No. S-9902; ULINE, CA) and shipped overnight via USPS in accordance withfederal regulations. Upon arrival, fecal sample were immediately storedat −80° C. This study was approved by the Committee on Human Research atthe University of California, San Francisco (CHR #10-03092). Physiciandiagnosed Ulcerative Colitis patients (age 18 to 60 years old) wererecruited directly from the gastroenterology clinic at UCSF's Mount ZionCampus. A questionnaire was provided to each patient to assess clinicalmeasures of disease severity [Simple Clinical Colitis Activity index(SCCA), extra-colonic manifestations (arthritis, pyoderma gangrenosum,erythema nodosum, and uveitis), number of first- and second-degreerelatives diagnosed with IBD, and duration of disease (years since UCdiagnosis)]. Healthy volunteers (age 18 to 60 years old) were drawn frompatients' families and by word of mouth. All participants wereself-reported to be of either European or South Asian ethnicity (FIG.37). Additionally, all participants resided within a 70-mile radius ofSan Francisco, Calif. Any participant experiencing pregnancy or breastfeeding, severe concomitant disease involving the liver, heart, lungs orkidneys, or antibiotic treatment within the preceding 2 months wereexcluded from the study.

Fecal DNA isolation. DNA was extracted from individual fecal samplesusing a combination of bead beating and the commercially availableQIAamp® DNA Stool Kit (Cat. No. 51504; QIAGEN, CA). Initially, 1.6 mL ofBuffer ASL was added to approximately 100 mg of feces and bead beat for30 s at 6.0 m/s in a FastPrep-24 instrument (Cat. No. 116004500; MPBiomedicals). Following bead beating, samples were incubated at 95° C.for 5 minutes to improve lysis efficiency of difficult to lyse microbes.The remainder of the DNA isolation was conducted using a QIAcube (Cat.No. 9001292; QIAGEN, CA) according to the QIAamp® DNA Stool KitProtocol: Isolation of DNA from Stool for Pathogen Detection. IsolatedDNA was stored at −80° C. Blank extractions were included as negativecontrols to monitor for bacterial contamination.

Bacterial 16S rRNA Gene Library Preparation. Bacterial 16S rRNA genesequencing libraries were created as previously described (52). PCRamplification of the 16S rRNA gene was conducted in triplicate for eachsample using barcoded primers targeting the V4 region as previouslydescribed (52). Blank extractions were used as template for negativecontrols to monitor for 16S rRNA contamination. PCR reactions wereperformed in 25 μl reactions using 0.025 U Takara Hot Start ExTaq(Takara Mirus Bio Inc, Madison, Wis.), 1× Takara buffer with MgCl₂, 0.4pmol μl⁻¹ of F515 and R806 primers, 0.56 mg ml⁻¹ of bovine serum albumin(BSA; Roche Applied Science, Indianapolis, Ind.), 200 μM of dNTPs, and10 ng of gDNA. Reactions were performed in triplicate under thefollowing conditions: initial denaturation (98° C. for 2 min) followedby 30 cycles of 98° C. (20 sec), annealing at 50° C. (30 sec), extensionat 72° C. (45 sec) and a final extension at 72° C. for 10 min. FollowingPCR, triplicates were pooled and 16s rRNA amplicon concentrations weredetermined via gel electrophoresis quantitation. 16S rRNA sequencelibrary was created by pooling all PCR amplicons in equimolarconcentrations to a final volume of 75 uL. To remove background, the 16SrRNA sequence library was run on a 2% agarose gel and the 16S amplicon(˜380 bp) was purified using the QIAquick Gel Extraction Kit (Cat. No.28704; QIAGEN, CA). The 16S rRNA primer sequences are provided inCaporaso J G, Lauber C L, Walters W A, Berg-Lyons D, Huntley J, FiererN, Owens S M, Betley J, Fraser L, Bauer M, Gormley N, Gilbert J A, SmithG, Knight R. 2012. Ultra-high-throughput microbial community analysis onthe Illumina HiSeq and MiSeq platforms. ISME J 6:1621-1624. Inembodiments, other primer sequences may be used.

Fungal ITS2 Library Preparation. Fungal internal transcribed spacer 2(ITS2) sequencing libraries were created using similar methods to thoseused for the 16S rRNA library. PCR amplification of the ITS2 region wasconducted in triplicate for each sample using barcoded primers. PCRreactions were performed in 25 μl reaction with 1× Takara buffer (TakaraMirus Bio), 200 nM of each primer, 200 μM dNTPs, 2.75 mM of MgCl₂, 0.56mg ml⁻¹ of BSA (Roche Applied Science), 0.025 U Takara Hot Start ExTaqand 50 ng of gDNA. Reactions were conducted under the followingconditions: initial denaturation (94° C. for 5 min) followed by 30cycles of 94° C. (30 sec), annealing at 54° C. (30 sec), extension at72° C. (30 sec) and a final extension at 72° C. for 7 min. FollowingPCR, triplicates were pooled and purified using the Agencourt AMPureXP-PCR Purification Kit and associated protocol (Cat. No. A63880,Beckman Coulter). Samples were quantified using the KAPA SYBR FAST qPCRKit (Cat. No. KK4601, KAPA Biosystems) as recommended by themanufacturers. All purified samples were then pooled in equimolarconcentrations based individual sample ITS2 quantification to a finalvolume of 75 uL.

16S and ITS2 Library Sequencing. Purified sequencing libraries wereanalyzed using a Bioanalyzer (Aligent), quantified using the Qubit HSdsDNA kit (Invitrogen), and diluted to 2 nM. Diluted sequence librarieswere then denatured, diluted to 5.88 pM, and combined with denatured12.5 pM PhiX spike-in to final concentration of 5 pM. Preparedsequencing libraries were then loaded onto the Illumina MiSeq cartridge(Cat. No. MS-102-3001, Illumina) and sequenced (514 cycles, Read 1: 251cycles, Index Read: 12 cycles, Read 2: 251 cycles) using a MiSeqplatform and MiSeq Control Software v2.2.0 according to themanufacturer's instructions (Illumina). All sequence data related tothis study is available in the Sequence Read Archive (SRA) database,ncbi.nlm.nih.gov/sra (accession no. PRJNA313074).

Bacterial 16S rRNA Sequence Processing.

Following paired-end sequencing, paired sequences were assembled usingFLASH v1.2.7 with a minimum overlap set at 15 bp (53). Assembled readswere de-multiplexed by barcode and filtered for low quality (Q-score<30)using QIIME 1.8 (54). If the Q-score three consecutive bases were <30,the read was truncated before the low-quality bases. The resulting readwas retained in the dataset if it was at least 75% of the originallength. Operational taxonomic units (OTUs) were picked at 97% sequenceidentity using uclust against the GreenGenes 13_8 database (55) (56),retaining OTUs containing >1 sequence read. Reads that failed to hit thereference sequence collection were retained and clustered de novo.Sequences were aligned using PyNAST and taxonomy was assigned usinguclust and the GreenGenes 13_8 database (57) (55) (56). PyNAST-alignedsequences were chimera checked using ChimeraSlayer (58), removingputative chimeras and representative sequences that failed PyNASTalignment. A phylogenetic tree was built using FastTree (59). Tonormalize variation in read depth across samples, data were rarefied tothe minimum read depth of 49,518 sequences per sample for bacteria. Toensure that a truly representative community of each sample was used foranalysis, sequence sub-sampling at the defined depth was bootstrapped100 times. The representative community composition for each sample wasdefined as that which exhibited the minimum average Canberra distance toall other OTU vectors generated from all sub-samplings for thatparticular sample.

Fungal ITS2 Sequence Processing.

Following paired-end sequencing, paired sequences were assembled usingFLASH v1.2.7 with a minimum overlap of 25 bp and a maximum overlap of290 bp (53). Assembled reads were de-multiplexed by barcode using QIIME1.8 (54). Assembled reads containing >2 expected errors, as determinedby usearch (55), were removed. Singleton reads were removed and OTUs of97% sequence similarity were generated de novo using usearch8.0 (55).The 8_1_2015 UNITE ITS fungal sequence database and usearch8.0 was usedto remove potentially chimeric sequences (60) (55). The ITSx softwarepackage was then used to extract the predicted ITS2 region from thereference sequence of non-chimeric OTUs, filtering out OTUs predicted tolack a true ITS2 region in the process (61). Taxonomy was then assignedto non-chimeric. ITS2 extracted OTUs using Bayesian classification witha confidence cut-off of 0.8 in QIIME according to the 8_1_2015 UNITE ITSfungal sequence database (54) (60). OTUs responsible for less that0.001% of the total sequence reads were removed. To normalize variationin read depth across samples, data were rarefied to the minimum readdepth of 6,653 sequences per sample for bacteria. To ensure that a trulyrepresentative community of each sample was used for analysis, sequencesub-sampling at the defined depth was bootstrapped 100 times. Therepresentative community composition for each sample was defined as thatwhich exhibited the minimum average Canberra distance to all other OTUvectors generated from all sub-samplings for that particular sample.

Bacterial 16S rRNA Gene Profiling Using PhyloChip. Total DNA extractedfrom fecal samples was used as template for 16S rRNA gene amplificationas previously described (62). PCR amplification was verified on a 1% TBEagarose gel then purified using the QIAquick Gel Extraction kit (Cat.No. 28704. QIAGEN, CA). A total of 500 ng of purified PCR product persample was then fragmented, biotin-labeled, and hybridized to the G3 16SrRNA PhyloChip (Affymetrix, CA) as previously described (63). Washing,staining, and scanning of arrays were conducted according to standardAffymetrix protocol (63). Background subtraction, detection, taxonquantification criteria and array normalization was performed aspreviously described (63). Stage 1 thresholds were adjusted, based onquantitative standards to the following: rQ1≥0.25, rQ2≥0.50, rQ3≥0.80.All PhyloChip microarray data reported in this paper has been depositedin the Gene Expression Omnibus (GEO) database, ncbi.nlm.nih.gov/geo(accession no. GSE78724).

Predicted community metagenome analyses. Phylogenetic Investigation ofCommunities by Reconstruction of Unobserved States (PICRUSt;picrust.github.io/picrust/), a bioinformatics software package used topredict functional metagenomes from a marker gene survey (such as 16SrRNA gene), was used to generate in silico bacterial metagenomes fordata generated in this study (64). First, the biom-formatted bacterialOTU table previously generated from the processed 16S rRNA gene MiSeqdata was filtered to contain only closed-reference OTUs [i.e. OTUspresent in the GreenGenes 16S rRNA 13_8 database (56)]. Theclosed-reference OTU table was then used to generate predictedmetagenomes according to the PICRUSt metagenome prediction tutorial(picrust.github.io/picrust/tutorials/metagenome_prediction.html-metagenome-prediction-tutorial).Briefly, OTU abundance was first normalized according to known orpredicted 16s copy number. Following 16s copy number normalization, thisnormalized OTU table was then used to predicted KEGG Ortholog (KO)abundances for each sample, which were further collapsed into KEGGPathways (genome.jp/kegg/pathway.html).

Metabolome Profiling. To profile fecal metabolites, >200 mg of frozenstool from each sample was shipped overnight on dry ice to Metabolon(Metabolon, NC). Also included were several technical replicate samplescreated from a homogeneous pool containing a small amount of all studysample. Upon receipt, samples were inventoried, and immediately storedat −80° C. At the time of analysis, samples were extracted and preparedfor analysis using Metabolon's standard solvent extraction method(metabolon.com/). The extracted samples were split into equal parts foranalysis on the GC/MS and Q-Exactive accurate mass LC/MS platforms.

Sample Preparation:

The sample preparation process was carried out using the automatedMicroLab STAR® system from Hamilton Company. Recovery standards wereadded prior to the first step in the extraction process for QC purposes.Sample preparation was conducted using a proprietary series of organicand aqueous extractions to remove the protein fraction while allowingmaximum recovery of small molecules. The resulting extract was dividedinto two fractions; one for analysis by LC/MS and one for analysis byGC/MS. Samples were placed briefly on a TurboVap® (Zymark) to remove theorganic solvent. Each sample was then frozen and dried under vacuum.Samples were then prepared for the appropriate instrument, either LC/MSor GC/MS.

QA/QC:

For QA/QC purposes, a number of additional samples were included witheach day's analysis. Furthermore, a selection of QC compounds was addedto every sample, including those under test. These compounds were chosenso as not to interfere with the measurement of the endogenous compounds.FIG. 38 and FIG. 39 describe the QC samples and compounds. These QCsamples are primarily used to evaluate the process control for eachstudy as well as aiding in the data curation.

Ultrahigh Performance Liquid Chromatography Mass Spectroscopy(UPLC/MS/MS):

The LC/MS portion of the platform was based on a Waters ACQUITYultra-performance liquid chromatography (UPLC) and a Thermo ScientificQ-Exactive high resolution/accurate mass spectrometer interfaced with aheated electrospray ionization (HESI-II) source and Orbitrap massanalyzer operated at 35,000 mass resolution. The sample extract wasdried then reconstituted in acidic or basic LC-compatible solvents, eachof which contained 8 or more injection standards at fixed concentrationsto ensure injection and chromatographic consistency. One aliquot wasanalyzed using acidic positive ion optimized conditions and the otherusing basic negative ion optimized conditions in two independentinjections using separate dedicated columns (Waters UPLC BEH C18-2.1×100mm, 1.7 μm). Extracts reconstituted in acidic conditions were gradienteluted using water and methanol containing 0.1% formic acid, while thebasic extracts, which also used water/methanol, contained 6.5 mMAmmonium Bicarbonate. The MS analysis alternated between MS anddata-dependent MS2 scans using dynamic exclusion, and the scan range wasfrom 80-1000 m/z. Raw data files are archived and extracted as describedbelow.

Gas chromatography Mass Spectrometry (GC/MS):

The samples destined for GC/MS analysis were re-dried under vacuumdesiccation for a minimum of 24 hours prior to being derivatized underdried nitrogen using bistrimethyl-silyl-triflouroacetamide (BSTFA). TheGC column was 5% phenyl/95% dimethyl polysiloxane fused silica columnand the temperature ramp was from 40° to 300° C. in a 16 minute period.Samples were analyzed on a Thermo-Finnigan Trace DSQ fast-scanningsingle-quadrupole mass spectrometer using electron impact ionization.The instrument was tuned and calibrated for mass resolution and massaccuracy on a daily basis. The information output from the raw datafiles was automatically extracted as discussed below.

Data Extraction and Compound Identification:

Raw data was extracted, peak-identified and QC processed usingMetabolon's hardware and software. Compounds were identified bycomparison to library entries of purified standards or recurrent unknownentities. Metabolon maintains a library based on authenticated standardscontaining the retention time/index (RI), mass to charge ratio (m/z),and chromatographic data (including MS/MS spectral data) on allmolecules present in the library. Furthermore, biochemicalidentifications are based on three criteria: retention index within anarrow RI window of the proposed identification, nominal mass match tothe library +/−0.4 amu, and the MS/MS forward and reverse scores betweenthe experimental data and authentic standards. The MS/MS scores arebased on a comparison of the ions present in the experimental spectrumto the ions present in the library spectrum.

Normalization:

For studies spanning multiple days, a data normalization step wasperformed to correct variation resulting from instrument inter-daytuning differences. Essentially, each compound was corrected in run-dayblocks by registering the medians to equal one (1.00) and normalizingeach data point proportionately. For studies that did not require morethan one day of analysis, no normalization was necessary.

In vitro DC/T-cell fecal water assay. Fecal Water Preparation. Fecalsamples were diluted in sterile 37° C. PBS containing 20% FBS and 2 mMEDTA to a final concentration of 1 g/mL. Diluted fecal samples were thenvortex for 1 minutes and incubated at 37° C. for 10 minutes. Followingincubation, samples were centrifuged at ˜21,000 g for 10 minutes at roomtemperature to remove insoluble material. Supernatants were thenfiltered through a 0.2 μm nylon filter to remove intact cells. Sterilefecal water solutions were stored at −20° C.

Dendritic Cell Fecal Water Challenge and T-Cell Co-Culture.

Peripheral blood samples were obtained from anonymous healthy humandonors (Blood Centers of the Pacific, San Francisco, Calif.). Peripheralblood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque gradientcentrifugation (Cat. No. Histopaque-10771; Sigma-Aldrich). Dendriticcells (DCs) were purified from isolated PBMCs using the EasySep™ HumanPan-DC Pre-Enrichment Kit (Cat. No. 19251; STEMCELL Technologies,Canada) and cultured in 96-well plates (0.5×10⁶ cells/ml) in fresh R10media: RPMI 1640 (Cat. No. 11875; Thermo-Fisher Scientific) supplementedwith 10% heat-inactivated FCS (Cat. No. 9871-5244; USA Scientific), 100U/ml penicillin-streptomycin (Cat. No. 10378016; Life Technologies, CA),10 ng/ml GM-CSF (Cat. No. 15-GM-010; R&D Systems, MN), and 20 ng/ml IL-4(Cat. No. 204-IL-010; R&D Systems). Prepared sterile fecal water wasadded to DC culture at a 1/20 dilution. After a 24 hour incubation,cells were stimulated with 10 ng/ml TNF-α (Cat. No. 300-01A; PeproTech,NJ), 10 ng/ml IL-1β (Cat. No. 200-01B; PeproTech), 10 ng/ml IL-6 (Cat.No. AF-200-06; PeproTech), and 1 μM PGE2 (Cat. No. 72194; STEMCELLTechnologies) and incubated for an additional 24 hours to induce DCmaturation. T-cells were purified from autologous, monocyte-depletedPBMCs by negative selection using the Human T-Cell Enrichment Column(Cat. No. HTCC-2000; R&D Systems) and were subsequently cultured inTexMACS Medium (Cat. Not. 130-097-196; Miltenyi Biotec, Germany).Following DC stimulation, DCs were harvested, washed, and co-culturedwith autologous T-cells at a ratio of 1/10 in the presence of 1 ug/mlsoluble anti-CD28 (Cat. No. 555725; BD Biosciences, CA) and 1 μg/mlanti-CD49d (Cat. No. 555501; BD Biosciences) for 5 days, replenishingthe media every 2 days. This assay was repeated four times using PBMCsobtained from distinct donors to ensure observations were independent ofPBMC source.

Flow Cytometry.

To assess cytokine production, the co-cultures were stimulated withPhorbol Myristate Acetate-lonomycin (Cat. No. 356150010; FisherScientific) and GolgiPlug (Cat. No. 555029; BD Biosciences) for 16hours. Cells were harvested and single-cell suspensions were stained intwo separate antibody panels to assess phenotype. Panel 1: anti-CD3(Cat. No. 557917, BD Biosciences), anti-CD4 (Cat. No. 563028; BDBiosciences), anti-CD8a (Cat. No. 563821; BioLegend), anti-CD25 (Cat.No. 557741; BD Biosciences), anti-FoxP3 (Cat. No. 14-4776-80;eBioscience), and anti-IL10 (Cat. No. 130-096-043; Miltenyi Biotec).Panel 2: anti-CD3 (Cat. No. 557917; BD Biosciences), anti-CD4 (Cat. No.563028; BD Biosciences), anti-CD8a (Cat. No. 563821; BioLegend),anti-CD69 (Cat. No. 560737; BD Biosciences), anti-INFγ (Cat. No. 560371;BD Biosciences), anti-IL4 (Cat. No. 130-091-647; Miltenyi Biotec),anti-IL117A (Cat. No. 17-7179-42; eBioscience), and anti-IL22 (Cat. No.25-7229-42; eBioscience). Cells were permeabilized by eitherCytofix/Cytoperm™ (Cat. No. 554714; BD Bioscience) orFixation/Permeabilization (Cat. No. 00-5523-00; Affymatrix eBioscience).Upon staining, live T-cells were gated as CD3⁺CD4⁺ or CD3⁺CD8⁺ cells.Activated T-cells were surface stained CD69hi. Among the CD4⁺ T-cellpopulation, subpopulations were defined as Th1: IFNγ⁺, Th2: IL-4⁺, Th17:IL-17A⁺, Th22: IL17A⁻ and IL-22⁺, and Treg: CD25hi and FoxP3hi. CD8⁺T-cells subpopulations were defined as Tc1: IFNγ⁺, Tc2: IL-4⁺, and Tc17:IL-17A⁺. Stained cells were assayed via flow cytometry on a BD LSR II(BD Biosciences).

Cytometric Bead Array.

Prior to addition of PMA/Gplug, 100 uL of cell-free supernatant wasremoved from each co-culture and centrifuged for 1 minute at 3000 rpm.Cytokine secretion was measured using a cytometric bead array (BDBiosciences) and the concentration of IL-4, IL-5, IL-13, and weredetermined according to the manufacturer's guidelines. Data was acquiredby flow cytometry on a BD LSR II (BD Biosciences) and data analysis wasperformed using the proprietary FCAP Array analysis software (BDBiosciences).

Statistical analysis. Microbial, Metagenomic, and Metabolomic Analysis.Analysis was performed using QIIME v1.8.0 and the R statisticalenvironment (54, 65). Shannon's Diversity and Faith's PhylogeneticDiversity were calculated using QIIME v 1.8.0 and two-tailed t-testswere performed to identify significant between group differences (e.g.UC vs. Healthy) (54). Weighted UniFrac, Canberra, and Bray-Curtisdistance matrices were generated using QIIME v 1.8.0 and visualized viaNMDS in the R statistical environment using the vegan package (66, 67).For PhyloChip data, fluorescent intensities were log-normalized prior tocalculating Canberra distances. Permutational multivariate analysis ofvariance (PERMANOVA) using calculated distance matrices was used todetermine relationships between existing metadata (i.e. Health Status orEthnicity) and bacterial, fungal, metagenome, or metabolome compositionusing the adonis function found in vegan (67). Hierarchical clusteranalysis combined with multi-scale, bootstrap resampling was performedusing the pvclust package in R with 1000 bootstrap replications (68).Correlation between distances matrices was calculated using the mantelfunction found in vegan (67). To identify significantly enriched ordepleted bacterial OTUs, fungal OTUs, and KEGG pathways between relevantsample groups (e.g. UC vs. Healthy), the three-model approach describedby Romero et al. was applied (69). Briefly, three linear mixed-effectregression models (negative binomial, zero-inflated negative bionomial,and Poisson) were independently fit to each observation (i.e. OTU orKEGG pathway) and the model with lowest Akaike Information Criterion(AIC) was retained. P-values were computed for only the best-fit models(i.e. those that minimized AIC). To account for false discovery,q-values were calculated based on the computed p-values. For PhyloChipdata, significantly enriched or depleted OTUs were determined byapplying a two-tailed t-test to log-normalized fluorescent intensities.To identify significantly enriched or depleted fecal metabolites,log-normalized relative concentrations were compared using a Welch'st-test.

Comparison of Clinical Measures of Disease Severity.

Clinical measures of disease severity (i.e. SCCA, number ofextra-colonic manifestations, number of diagnosed first- andsecond-degree relatives, and years since diagnosis were compared betweenUC-MCS by a Kruskal-Wallis Test followed by pairwise tw-tailed Dun'sTest.

Analysis of T-Cell Subsets.

Because the T-cell assay described above was repeated four separatetimes using PBMCs from four different PBMC donors, a linear mixedeffects model was applied using the lme4 package in R to identifysignificant differences in the abundance of induced T-cellsubpopulations based on sample group (i.e. UC-MCS) while accounting forpotential variation introduced due to PBMC source (i.e. donor) (70). Thefollowing linear mixed effects models were applied to identify changesdue to health status (Healthy vs. UC) and UC-MCS (Healthy vs. MCS1,MCS2, MCS3, MCS4) respectively:Y˜β((EXP_GROUP)+μ(DONOR)+μ(SAMPLE)+εY˜β(MCS)+μ(DONOR)+μ(SAMPLE)+εWhere Y=a measured, dependent variable such as Th1 abundance,EXP_GROUP=health status (Healthy or UC), MCS=microbial community state(Healthy, MCS1, MCS2, MCS3, or MCS4), DONOR=PBMC donor source (Donor #1to #4), and SAMPLE=fecal sample study participant.

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What is claimed is:
 1. A method of treating or reducing the incidence ofallergic inflammation, the method comprising administering a bacterialpopulation comprising isolates of at least: Lactobacillus sp.,Faecalibacterium sp., and Akkermansia muciniphila to a subject in needthereof, wherein the bacterial population is present in an effectiveamount for treating or reducing the incidence of allergic inflammationin the subject.
 2. The method of claim 1, wherein the Lactobacillus sp.is Lactobacillus johnsonii.
 3. The method of claim 1, wherein theFaecalibacterium sp. is Faecalibacterium prausnitzii.
 4. The method ofclaim 1, wherein the Lactobacillus sp. is Lactobacillus zeae,Lactobacillus acidipiscis, Lactobacillus acidophilus, Lactobacillusagilis, Lactobacillus aviarius, Lactobacillus brevis, Lactobacilluscoleohominis, Lactobacillus crispatus, Lactobacillus crustorum,Lactobacillus curvatus, Lactobacillus diolivorans, Lactobacillusfarraginis, Lactobacillus fermentum, Lactobacillus fuchuensis,Lactobacillus harbinensis, Lactobacillus helveticus, Lactobacillushilgardii, Lactobacillus intestinalis, Lactobacillus jensenii,Lactobacillus kefiranofaciens, Lactobacillus kefiri, Lactobacilluslindneri, Lactobacillus mali, Lactobacillus manihotivorans,Lactobacillus mucosae, Lactobacillus oeni, Lactobacillusoligofermentans, Lactobacillus panis, Lactobacillus pantheris,Lactobacillus parabrevis, Lactobacillus paracollinoides, Lactobacillusparakefiri, Lactobacillus paraplantarum, Lactobacillus pentosus,Lactobacillus pontis, Lactobacillus reuteri, Lactobacillus rossiae,Lactobacillus salivarius, Lactobacillus siliginis, Lactobacillussucicola, Lactobacillus vaccinostercus, Lactobacillus vaginalis,Lactobacillus vini, Lactococcus garvieae, or Lactococcus lactis; andwherein the Faecalibacterium sp., is Faecalibacterium prausnitzii. 5.The method of claim 1, wherein the bacterial population furthercomprises Bifidobacterium sp.
 6. The method of claim 5, wherein theBifidobacterium sp. is Bifidobacterium bifidum, Bifidobacteriumpseudolongum, Bifidobacterium saeculare, or Bifidobacterium subtile. 7.The method of claim 1, wherein the bacterial population comprises lessthan 20 species of bacteria.
 8. The method of claim 1, wherein thebacterial population is part of a bacterial composition that is not afecal transplant.
 9. The method of claim 1, wherein the bacterialpopulation is part of a bacterial composition that comprises apharmaceutically acceptable excipient.
 10. The method of claim 1,wherein the bacterial population is administered orally or rectally. 11.The method of claim 1, wherein the allergic inflammation is ahypersensitivity, pediatric allergic asthma, allergic asthma, foodallergy, or atopic dermatitis.
 12. The method of claim 1, wherein theallergic inflammation is pediatric allergic asthma or inflammatory boweldisease.
 13. The method of claim 1, wherein the subject has at least 1relative who has been diagnosed with an inflammatory disease.
 14. Themethod of claim 1, wherein the subject suffers from constipation,diarrhea, bloating, urgency, or abdominal pain.
 15. The method of claim1, wherein the subject has been administered an antibiotic within thelast 1 month.
 16. The method of claim 1, wherein the subject is aneonate.
 17. The method of claim 1, wherein the subject is less thanabout 24 months old.
 18. The method of claim 1, wherein the subject isbetween about 2 and about 18 years old.
 19. The method of claim 1,wherein the subject comprises a gastrointestinal microbiome that (a) hasan increased proportion of Streptococcus spp., and Enterococcus spp.compared to a healthy or general population; (b) has a reducedproportion of Alternaria alternata, Aspergillus flavus, Aspergilluscibarius, and Candida sojae compared to a healthy or general population;(c) has an increased proportion of Candida albicans and Debaryomycesspp. compared to a healthy or general population; (d) has a reducedproportion of Bifidobacteria spp., Lactobacillus spp., Faecalibacteriumspp. and Akkermansia spp. compared to a healthy or general population;(e) has a reduced proportion of Malassezia spp. compared to a healthy orgeneral population; (f) has an increased proportion of Bacterioides spp.or Prevotella spp. compared to a healthy or general population; or (g)has an increased proportion of Enterococcus fiaecalis, Enterococcusfaecium, or Clostridium difficile compared to a healthy or generalpopulation.
 20. The method of claim 1, further comprising administeringa Cystobacter sp. or a fungal microorganism to the subject.
 21. Themethod of claim 1, wherein the bacterial population is part of abacterial composition that is lyophilized.
 22. The method of claim 1,wherein the bacterial population is part of a bacterial composition thatis in liquid form.
 23. The method of claim 1, wherein the inflammationis chronic inflammation.
 24. The method of claim 1, wherein theLactobacillus sp. is Lactobacillus crispatus, and wherein theFaecalibacterium sp., is Faecalibacterium prausnitzii.
 25. The method ofclaim 24, wherein the bacterial population consists of three bacterialspecies.
 26. The method of claim 1, wherein the Lactobacillus sp. isLactobacillus johnsonii, and wherein the Faecalibacterium sp., isFaecalibacterium prausnitzii.
 27. The method of claim 26, wherein thebacterial population consists of three bacteria species.